https://ivaprime.com/about/

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30                        <div class="about-header">
31                            <img src="/static/assets/IVA-Prime-Logo.png" width="300px">
32                            <div class="about-header-title">
33                                IVA Prime
34                            </div>
35                        </div>
36
37                        <!-- About IVA Prime -->
38                        <div class="about-section" id="about">
39                            <div class="about-section-title">
40                                About IVA Prime
41                            </div>
42                            <p>
43                                IVA Prime is a web application that allows you to import plasmid files (.gb and .dna), and then easily generate primers for IVA cloning as you edit your plasmid file. The primers can be then easily exported to a read-to-order format and the altered plasmid file can be saved.
44                            </p>
45                            <p>
46                                The primer design technique is originally based on the work by García-Nafría et al.: <a href="https://www.nature.com/articles/srep27459" target="_blank" class="underlined-link">DOI: 10.1038/srep27459</a>.
47                            </p>
48                            <p>
49                                The source code for IVA Prime can be found in the <a href="https://github.com/RaduLeonte/IVA-Prime" target="_blank" class="underlined-link">GitHub repository</a>.
50                            </p>
51                            <p>
52                                IVA Prime is described in detail in our recent publication: <a href="https://doi.org/10.1093/nar/gkaf386" target="_blank" class="underlined-link underlined-link-orange">DOI: 10.1093/nar/gkaf386</a>.
53                            </p>
54                        </div>
55
56                        <!-- How to use -->
57                        <div class="about-section" id="how-to-use">
58                            <div class="about-section-title">
59                                How to use
60                            </div>
61                            <div class="about-section-subtitle">
62                                Importing
63                            </div>
64                            <ul>
65                                <li>
66                                    <p>
67                                        Plasmid files can be imported by clicking on
68                                        <span class="button-about button-round button-green">Import File</span> to import a
69                                        .gb or .dna file. Files can also be imported by dragging files over the page and
70                                        then dropping them by releasing the mouse.
71                                    </p>
72                                </li>
73                                <li>
74                                    <p>
75                                        New files can be created by clicking on <span class="button-about button-round button-green">New File</span>
76                                        and specifying a DNA sequence. By default, common features will be automatically annotated, but can be turned
77                                        off. The topology of the pasted sequence can also be specified.
78                                    </p>
79                                </li>
80                                <li>
81                                    <p>
82                                        A common pET-28a(+) vector can be imported by clicking on
83                                        <span class="button-about button-round button-orange">Demo File</span>.
84                                    </p>
85                                </li>
86                            </ul>
87
88                            <div class="about-section-subtitle">
89                                Insertions
90                            </div>
91                            <ol>
92                                <li>
93                                    <p>
94                                        Left click at the position you would like to insert a sequence to set the sequence cursor.
95                                    </p>
96
97                                    <!-- <p>
98                                        <img src="/static/assets/gifs/insertion%20position%20selection.gif">
99                                    </p> -->
100                                </li>
101
102                                <li>
103                                    <p>
104                                        Right-click to open the context menu and select "Insert here".
105                                    </p>
106
107                                    <!-- <p>
108                                        <img src="/static/assets/gifs/insertion%20menu%20item%20selection.gif">
109                                    </p> -->
110                                </li>
111
112                                <li>
113                                    <p>
114                                        Enter a DNA or amino acid sequence to insert.
115                                    </p>
116                                </li>
117
118                                <li>
119                                    <p>
120                                        Optionally, select a target organism for codon optimisation (Escherichia coli, human, yeast etc.).
121                                    </p>
122                                </li>
123
124                                <li>
125                                    <p>
126                                        If you wish for the insertion to be translated, check the "Translate new feature" 

126checkbox.
127                                    </p>
128                                </li>
129
130                                <li>
131                                    <p>
132                                        Press <span class="button-about button-round button-green">Create primers</span>.
133                                    </p>
134                                </li>
135                            </ol>
136
137                            <div class="about-section-subtitle">
138                                Deletions
139                            </div>
140                            <ol>
141                                <li>
142                                    <p>
143                                        Select a region to be deleted by clicking and then draggin the mouse to set the selection cursors.
144                                    </p>
145                                    <!-- <p>
146                                        <img src="/static/assets/gifs/deletion%20selection.gif" alt="Selecting region to be deleted.">
147                                    </p> -->
148                                </li>
149                                <li>
150                                    <p>
151                                        Right-click to open the context menu and select "Delete selection".
152                                    </p>
153                                    <!-- <p>
154                                        <img src="/static/assets/gifs/deletion%20menu%20item%20selection.gif">
155                                    </p> -->
156                                </li>
157                            </ol>
158
159
160                            <div class="about-section-subtitle">
161                                Mutations
162                            </div>
163                            <ol>
164                                <li>
165                                    <p>
166                                        Select a region to be mutated by clicking and then draggin the mouse to set the selection cursors..
167                                    </p>
168                                    <!-- <p>
169                                        <img src="/static/assets/gifs/mutation%20selection.gif">
170                                    </p> -->
171                                </li>
172                                <li>
173                                    <p>
174                                        Right-click to open the context menu and select "Mutate selection".
175                                    </p>
176                                    <!-- <p>
177                                        <img src="/static/assets/gifs/mutation%20menu%20item%20selection.gif">
178                                    </p> -->
179                                </li>
180                                <li>
181                                    <p>
182                                        Enter a DNA or amino acid sequence.
183                                    </p>
184                                </li>
185                                <li>
186                                    <p>
187                                        Optionally, select a target organism for codon optimisation (Escherichia coli, human, yeast etc.).
188                                    </p>
189                                </li>
190                                <li>
191                                    <p>
192                                        If you wish for the insertion to be translated, check the "Translate new feature" checkbox.
193                                    </p>
194                                </li>
195                                <li>
196                                    <p>
197                                        Press <span class="button-about button-round button-green">Create primers</span>.
198                                    </p>
199                                </li>
200                            </ol>
201
202
203                            <div class="about-section-subtitle">
204                                Subcloning
205                            </div>
206                            <ol>
207                                <li>
208                                    <p>
209                                        To subclone navigate to your region of interest and select it.
210                                    </p>
211                                    <!-- <p>
212                                        <img src="/static/assets/gifs/subcloning%20selection.gif">
213                                    </p> -->
214                                </li>
215
216                                <li>
217                                    <p>
218                                        Right-click to open the context menu and select "Mark selection for subcloning". The sequence
219                                        will  then be highlighted, confirming the region as marked.
220                                    </p>
221                                </li>
222
223                                <li>
224                                    <p>
225                                        Now switch to the destination plasmid and navigate to where the marked sequence will be inserted.
226                                    </p>
227                                </li>
228
229                                <li>
230                                    <p>
231                                        To subclone between two base

231s, left-click at desired position to set the cursor, then right-click 
232                                        and choose "Subclone into selection". Alternatively, if you wish to replace a region with the subcloning
233                                        target, select the region and then right-click and select "Subclone into selection".
234                                    </p>
235                                </li>
236
237                                <li>
238                                    <p>
239                                        Optioally, if you wish to at the same time insert additional sequences adjacent to the subcloning target 
240                                        sequence at the destination, choose "Subclone with insertion into selection" in the context menu. 
241                                        This will open a window where you can specify the additional sequences that will be included at the 
242                                        5' and 3' prime ends individually. Once you are done, press 
243                                        <span class="button-about button-round button-green">Create primers</span>.
244                                    </p>
245                                </li>
246                            </ol>
247
248                            <div class="about-section-subtitle" id="lin-frag">
249                                Insertions from linear fragments
250                            </div>
251
252                            <p>
253                                Insertions from linear fragments works similarly to subcloning. During subcloning, we create a linear fragment of the 
254                                subcloning target with overhangs that will be homologous to the target vector using the insert primers and also linearize 
255                                the target vector using the vector primers. However, if one needs to order a gene from a supplier, the former reaction can 
256                                be skipped by ordering the gene with the appropriate homologous overhangs. 
257                            </p>
258
259                            <p>
260                                IVA Prime can generate the sequence of the linear fragment with the overhangs and also the primers needed to linearize the 
261                                target vector.
262                            </p>
263
264                            <ol>
265                                <li>
266                                    <p>
267                                        Left click at the position you would like to insert a sequence to set the sequence cursor or select a range that 
268                                        should be replaced with your insertion.
269                                    </p>
270                                </li>
271
272                                <li>
273                                    <p>
274                                        Right-click to open the context menu and select "Insert from linear fragment".
275                                    </p>
276                                </li>
277
278                                <li>
279                                    <p>
280                                        Optionally, give a name to your linear fragment.
281                                    </p>
282                                </li>
283
284                                <li>
285                                    <p>
286                                        Enter a DNA or amino acid sequence to insert.
287                                    </p>
288                                </li>
289
290                                <li>
291                                    <p>
292                                        Optionally, select a target organism for codon optimisation (Escherichia coli, human, yeast etc.).
293                                    </p>
294                                </li>
295
296                                <li>
297                                    <p>
298                                        If you wish for the insertion to be translated, check the "Translate new feature" checkbox.
299                                    </p>
300                                </li>
301
302                                <li>
303                                    <p>
304                                        Press <span class="button-about button-round button-green">Create primers and linear fragment</span>.
305                                    </p>
306                                </li>
307
308                                <li>
309                                    <p>
310                                        The generated vector primers will be displayed in the sidebar of the target vector and the newly generated
311                                        linear fragment file will be available in a new tab.
312                                    </p>
313                                </li>
314                            </ol>
315
316
317                            <div class="about-section-subtitle">
318                                Saving the primers and modified plasmid to file
319                            </div>
320                            <p>
321                                Primers and the modified plasmid file can be exported to a variety of formats by
322                                clicking on the downwards pointing arrow on the tab of your plasmid. This will open
323                                a dropdown menu where you may select the output format.
324                            </p>
325                        </div>
326
327                        <div class="about-section">
328                            <div class="about-section-title">
329                                IVA cloning protocol
330                            </div>
331
332                            <div class="about-section-subtitle">
333                                Reaction setup
334                            </div>
335
336                            <p>
337                                Non-subcloning reactions:
338                            </p>
339                            <ul>
340                                <li>
341                                    <p>
342                                        1 &mu;L primer pair mix (forward and reverse primers mixed at 2.5 &mu;M)
343                                    </p>
344                                </li>
345                                <li>
346                                    <p>
347                                        1 &mu;L diluted template plasmid (1 ng/&mu;L)
348                                    </p>
349                                </li>
350                                <li>
351                                    <p>
352                                        12.5 &mu;L Phusion High-Fidelity PCR Master Mix (HF Buffer, Thermo Fisher Scientific)
353                                    </p>
354                                </li>
355                                <li>
356                                    <p>
357                                        10.5 &mu;L ddH<sub>2</sub>O
358                                    </p>
359                                </li>
360                            </ul>
361
362                            <p>
363                                Subcloning reactions:
364                            </p>
365                            <ul>
366                                <li>
367                                    <p>
368                                        1 &mu;L insert primer pair mix (forward and reverse primers mixed at 2.5 &mu;M)
369                                    </p>
370                                </li>
371                                <li>
372                                    <p>
373                                        1 &mu;L vector primer pair mix (vector forward and vector reverse primers mixed at 2.5 &mu;M)
374                                    </p>
375                                </li>
376                                <li>
377                                    <p>
378                                        1 &mu;L diluted template plasmid (1 ng/&mu;L)
379                                    </p>
380                                </li>
381                                <li>
382                                    <p>
383                                        1 &mu;L diluted source plasmid (1 ng/&mu;L)
384                                    </p>
385                                </li>
386                                <li>
387                                    <p>
388                                        12.5 &mu;L Phusion High-Fidelity PCR Master Mix (HF Buffer, Thermo Fisher Scientific)
389                                    </p>
390                                </li>
391                                <li>
392                                    <p>
393                                        8.5 &mu;L ddH<sub>2</sub>O
394                                    </p>
395                                </li>
396                            </ul>
397
398                            <div class="about-section-subtitle">
399                                PCR program
400                            </div>
401
402                            <ol>
403                                <li>
404                                    <p>
405                                        98 °C for 30 s (initial denaturation)
406                                    </p>
407                                </li>
408                                <li>
409                                    <p>
410                                        98 °C for 10 s (denaturation)
411                                    </p>
412                                </li>
413                                <li>
414                                    <p>
415                                        60 °C for 30 s (annealing)
416                                    </p>
417                                </li>
418                                <li>
419                                    <p>
420                                        72 °C for 30 s (extension)
421                                    </p>
422                                </li>
423                                <li>
424                                    <p>
425                                        Go to step nr. 2 (29 times)
426                                    </p>
427                                </li>
428                                <li>
429                                    <p>
430                                        72 °C for 5 min (final extension)
431                                    </p>
432                                </li>
433                            </ol>
434
435                            <div class="about-section-subtitle">
436                                Transformation
437                            </div>
438                            
439                            <ol>
440                                <li>
441                                    <p>
442                                        Digest PCR product with 0.5 DpnI (10 U/&mu;L) for 30 min at 37 °C. 
443                                    </p>
444                                </li>
445                                <li>
446                                    <p>
447                                        Add 2 &mu;L of the PCR product to 48 &mu;L KCM (500 mM KCl, 150 mM CaCl<sub>2</sub>, 250 mM MgCl<sub>2</sub>).
448                                    </p>
449                                </li>
450                                <li>
451                                    <p>
452                                        Add mixture to 50 &mu;L competent <i>E. coli</i>.
453                                    </p>
454                                </li>
455                                <li>
456                                    <p>
457                                        Incubate on ice for 30 min.
458                                    </p>
459                                </li>
460                                <li>
461                                    <p>
462                                        Add 900 &mu;L SOC media.
463                                    </p>
464                                </li>
465                                <li>
466                                    <p>
467                                        Incubate for 1 hour at 37 °C.
468                                    </p>
469                                </li>
470                                <li>
471                                    <p>
472                                        Non-subcloning operations: plate 100 &mu;L.
473                                    </p>
474                                    <p>
475                                        Subcloning operations: spin-down bacteria suspension, resuspend pellet in 50 &mu;L SOC media and plate.
476                                    </p>
477                                </li>
478                            </ol>
479
480                        </div>
481
482                        <div class="about-section">
483                            <div class="about-section-title">
484                                IVA Prime settings
485                            </div>
486
487                            <div class="about-section-subtitle">
488                                Basic settings
489                            </div>
490
491                            <ul>
492                                <li>
493                                    <p>
494                                        <b>Primer type:</b> Asymmetric (Asym.) / Symmetric (Sym.)
495                                    </p>
496                                    <p>
497                                        Asymmetric primers will have their Homologous Region (HR) distributed to only the forward 
498                                        primer, while symmetric primers will have their HR distributed evenly amongst the forward 
499                                        and reverse primer. Asymmetric primers are useful when doing many operations starting at 
500                                        the same position, as that will allow the reuse of the reverse primer. Symmetric primers 
501                                        have their forward and reverse primer lengths averaged and thus can save costs depending 
502                                        on the pricing of your local oligo supplier.
503                                    </p>
504                                </li>
505                                <li>
506                                    <p>
507                                        <b>Theme:</b> Light / Dark
508                                    </p>
509                                    <p>
510                                        IVA Prime offers an alternative theme with the dark theme for users who want to reduce 
511                                        eye strain.
512                                    </p>
513                                </li>
514                            </ul>
515                            
516                            <div class="about-section-subtitle">
517                                Advanced settings
518                            </div>
519
520                            <ul>
521                                <li>
522                                    <p>
523                                        <b>Primer concentration</b>
524                                    </p>
525                                    <p>
526                                        The primer concentration in nM specifies the amount of DNA for the purposes of c

526alculating
527                                        the Tm. This value is only relevant for the Nearest-neighbor algorithm.
528                                    </p>
529                                </li>
530                                <li>
531                                    <p>
532                                        <b>Algorithm:</b> Basic (Oligo Calc) / Nearest-neighbor (SantaLucia)
533                                    </p>
534                                    <p>
535                                        This setting specifies the algorithm that is to be used for calculting the melting temperatures
536                                        of DNA sequences. The Oligo Calc algorithm (<a href="https://doi.org/10.1093/nar/gkm234" target="_blank">DOI: 10.1093/nar/gkm234</a>) 
537                                        is quite basic but has proven robust for IVA Clonig. 
538                                        If a more realistic algorithm is desired, the Nearest-neihbour algorithm by SantaLucia (<a href="https://doi.org/10.1021/bi951907q" target="_blank">DOI: 10.1021/bi951907q</a>) can be chosen. 
539                                        This setting only affects the primer generation of the template binding regions 
540                                        (TBRs) as the generation of the homologous regions (HRs) have been optimized previously using the 
541                                        Oligo Calc algorithm. The exact equation of the selected algorithm is displayed in the settings panel.
542                                    </p>
543                                </li>
544                                <li>
545                                    <p>
546                                        <b>Salt concentration</b>
547                                    </p>
548                                    <p>
549                                        The concentration of salt in M for the purposes of c

549alculating the Tm correction due to ions interacting
550                                        with the DNA.
551                                    </p>
552                                </li>
553                                <li>
554                                    <p>
555                                        <b>Salt correction: </b> Schildkraut-Lifson / Owczarzy
556                                    </p>
557                                    <p>
558                                        The method for the salt correction can be specified here. Two common salt correction methods are offered:
559                                        the correction by Shildkraut and Lifson (<a href="https://doi.org/10.1002/bip.360030207" target="_blank">DOI: 10.1002/bip.360030207</a>), which depends solely on the concentration of ions, or the higher 
560                                        order correction by Owczarzy (<a href="https://doi.org/10.1021/bi034621r" target="_blank">DOI: 10.1021/bi034621r</a>), which includes the GC fractional content of the sequence. The exact equation 
561                                        of the selected salt correction is displayed in the settings panel.
562                                    </p>
563                                </li>
564                                <li>
565                                    <p>
566                                        <b>DMSO concentration</b>
567                                    </p>
568                                    <p>
569                                        The concentration of DMSO in M used in the DMSO correction (equation displayed in the settings panel).
570                                    </p>
571                                </li>
572                                <li>
573                                    <p>
574                                        <b>HR minimum length</b>
575                                    </p>
576                                    <p>
577                                        Minimum length in bp of the homologous region (HR). In cases of high GC content, the primers may reach their
578                                        target melting temperatuer Tm with very short sequences. However, very short primer sequences have lower
579                                        recombination efficiency and may therefore fail. Thus, by default the HRs have a minimum length, but this 
580                                        may be turned off.
581                                    </p>
582                                </li>
583                                <li>
584                                    <p>
585                                        <b>HR target Tm</b>
586                                    </p>
587                                    <p>
588                                        The target melting temperature (Tm) of the homologous region (HR). This setting specifies the value for the Tm
589                                        of the HR sequence that should be achieved when extending the primer sequence for non-subcloning operations.
590                                    </p>
591                                </li>
592                                <li>
593                                    <p>
594                                        <b>Subcloning HR target Tm</b>
595                                    </p>
596                                    <p>
597                                        The target melting temperature (Tm) of the homologous region (HR) for subcloning operations. Subcloning operations
598                                        have lower recombination efficiencies due to the increased number of recombination events (2) needed when compared 
599                                        to the basic operations (Insertions, deletions, mutations; 1 recombination event). Therefore, the target Tm that
600                                        the HRs must reach when extending the primer sequences is set by default to a higher value than for the basic 
601                                        operations, but this may changed if so desired.
602                                    </p>
603                                </li>
604                                <li>
605                                    <p>
606                                        <b>TBR target tm</b>
607                                    </p>
608                                    <p>
609                                        The target melting temperatuer (Tm) for the template binding region (TBR), which linearize the plasmid. When extending
610                                        the primer sequence, the Tm algorithm specified in the "Algorithm" setting will be used to determine the Tm of the primer. 
611                                    </p>
612                                </li>
613                                <li>
614                                    <p>
615                                        <b>Maximum Tm for short insertions</b>
616                                    </p>
617                                    <p>
618                                        The maximum melting temperature (Tm) for short insertions. When generating asymmetric primers (see "Primer type" setting),
619                                        the homologous region (HR), which in the case of insertions or mutations will contain the new sequence, will be distributed 
620                                        only on the forward primer. However, for long insertions, which contain new sequences longer than what the HR needs to be,
621                                        the entire new sequence will be on the forward primer and only a shorter part (enough to server as the HR) will be on the 
622                                        reverse primer. This setting specifies the maximum Tm that new sequences can have and still generate short insertion primers. 
623                                    </p>
624                                </li>
625                                <li>
626                                    <p>
627                                        <b>Use GC clamp</b>
628                                    </p>
629                                    <p>
630                                        The GC clamp in primer design ensures that the primer sequence starts (5' end) or ends (3' end) with a G or C base. This approach is used to 
631                                        increase the stability of the primer's binding due to the stronger G-C pair, compared to the A-T pair. By incorporating a GC 
632                                        clamp, the primer is more likely to initiate annealing at this position. This setting allows you to enable or disable the use 
633                                        of the GC clamp.  
634                                    </p>
635                                </li>
636                            </ul>
637
638                        </div>
639
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641                        <!-- Privacy policy -->
642                        <div class="about-section" id="privacy-policy">
643                            <div class="about-section-title">
644                                Privacy policy
645                            </div>
646                            <p>IVA Prime uses first-party cookies to improve the user experience by storing user preferences, 
647                                such as preferred display options or algorithm settings, between sessions. No third-party 
648                                cookies are used to track or profile users.</p>
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