Hi Shahid

If you are doing site directed mutagenesis of a whole plasmid with a pair of complementary primers that contain the mutation, as in QuickChange method, THAT IS NOT PCR: there is no exponential amplification with the DNA polymerase, so you´re not going to see in agarose gel any amplified band as in a standard PCR (your initial amount of DNA for the mutagenesis reaction could be 5-20 ng). Just go ahead with the DpnI digestion and transform good competent cells. I usually get more than 95 % of mutants when using QuickChange.

Good luck.

Hi Shahid,

I guess you are doing whole plasmid PCR using the quickchange kit originally introduced by Stratagene. In that case your cycling conditions are absolutely ok. Efficiency of this method drops with increasing template size and 9kb is pretty large but should work. One thing I do to optimize my mutagensis PCR is playing around with template concenrations. I usually dilute a standard plasmid prep 1:10 and 1:100 and use 1 ul each in two indenpendent mutagensis PCR runs, sometimes only one concentration works, sometimes both. Another thing that appears critical using large templates is the qualitiy of your plasmid prep. In you case it might be worthwhile preparing a really clean prep using a kit.

Even if you don't see a band after you've run the PCR there might still be enough product to obtain colonies after transformation. You could just be confident, do the DpnI digest and use highly competent cells for transformation. Maybe you are lucky.

Cheers,

Ricky

Actually I could see a very very faint band if I try to contrast my gel picture and it correspond to the same kbp of ladder DNA as expected . This means should I go ahead with Dpn1 digestion and transformation?

Yes, its definetly worth a try.

@Shahid: Yes, go ahead. I usually never checked site-directed mutagenesis on a gel but simply added the DpnI to the PCR mix. As Frederik said, the efficiency is quite low, as is the number of cycles. After inactivation we usually transformed different amount into E.coli (or whatever bacteria you are using) and checked the positive clones. There we prepared a few and sequenced the region in question.

@Andrei/Gabriele: Shahid is trying to do site-directed mutagenesis, the PCR protocol for this is quite different from standard PCR.

Thank you very much for the heads-up and I will go tomorrow and perform the Dpn1 digestion and transformation. Thank you very much once again

Hi Shahid

If you are doing site directed mutagenesis of a whole plasmid with a pair of complementary primers that contain the mutation, as in QuickChange method, THAT IS NOT PCR: there is no exponential amplification with the DNA polymerase, so you´re not going to see in agarose gel any amplified band as in a standard PCR (your initial amount of DNA for the mutagenesis reaction could be 5-20 ng). Just go ahead with the DpnI digestion and transform good competent cells. I usually get more than 95 % of mutants when using QuickChange.

Good luck.

I have done SDM a few times and have never seen a band on a gel. If you are using a proof-reading DNA polymerase, I think Phusion is one, Make sure the mutation you are trying to introduce is buried well within your primers. However, the primers do not need to be as long as Stratagene's Primer Design tool makes them, 30 to 40-mers. I have had success with 20 to 25-mers. Just make sure your mutation is 10 nt or more from the 3-prime end of your primers. Use as little template as possible to reduce background, and I often incubate Dpn I digests overnight at 37 C. I usually use the Quick Change kit from Stratagene (Agilent) but other kits should work as well.

I've done Site directed mutagenesis quite a few times, managed exchange ~12 nucleotides in single step, remove/add, anything is possible.

There are different protocols, I use the one with complementary primers.

As for the primer size, I always make sure that the annealing temperature of the fragments left and right from the mutation site is around 50°C. As José stated, it is not a PCR so observing the band on the gel is not mandatory, I do transformation directly after DpnI dogestion.

Now, people have suggested using Taq polymerase. I have heard it works for some but it never worked for me. Taq polymerase have strand displacement which is really undesirable for SDM.

50 uL reaction contains:

5 uL Buffer

1 uL dNTPs (10 mM)

2x 1.5 uL Primers (each 10 uM)

50 ng of plasmid

1 uL Pfu Polymerase

water up to 50.

I also prepare reactions with 5 % DMSO, in practice I just prepare one 50 uL reaction, aliquote into two 25 uL and in one of the aliquotes add 1.25 uL DMSO (the volume change does not influence the overall reaction). Then I separate each 25 uL into two 12.5 uL (thus having total 4x 12.5 uL) and run at two different temperatures, with or without DMSO.

Temperature program:

95 °C 5 mins

95 °C 45 s

50 or 59 °C 45s (annealing

68 °C (2 mins per 1kbp) back to step 2 (15 cycles total)

68 °C 10 mins

After this I add DpnI for 2 to 4 hours and then directly transform. If I see colonies, I usually have 90 % success rate, unless there was something wrong with the DpnI digestion.

Andrei, it is not PCR, the increase of product is linear, not exponential.

@Andrei: as Frederik and Jiri said before, it seems Shahid is using QuickChange method to generate the mutation. The use of a DNA polymerase and temperature cycling doesn´t mean it´s a PCR. There is no exponential amplification (after every cycle, the amount of DNA product is not doubled), so it´s not PCR.

@Andrei: No offense, I was just saying. Its not an exponential amplification though, since you "only" change the plasmids present in the reaction.

@Andrei:

The question by Shahid was: "I couldn't see any band in the gel. Do you know what could be the problem?" The answer could be : "there is no problem. There is no exponential amplification -so, there is no strong band in gel- because it´s not a PCR reaction".

Exponential amplification is one of the features that define the PCR. But if your concept of PCR includes a linear amplification reaction, then it´s ok.

A clear explanation by Kary Mullis:

http://www.dnalc.org/view/15140-Making-many-DNA-copies-Kary-Mullis.html

Thank you very much everyone for the reply. I did the Dpn1 digestion of the PCR product and have transformed the plasmid in highly competent E coli cells. Will see tomorrow if I have any colonies or not. If I get the colonies, I will send the sample for DNA sequencing.

I think the annealing temperature you are using is too high...If I were you I would try 52ºC and see if you have band...if you get more than 1 band you can try to rise the temperature, but not up to 68

Try different primer designing method as described http://www.biomedcentral.com/1472-6750/8/91

Such a long extension time should not be necessary. Pfu Turbo has a rapid extension time, although I am uncertain of the exact value. Phusion is roughly 2-2.5 kbp/min. NEB's newer polymerase, Q5, has about the same extension rate but is even more robust than Phusion. Both have 3'-5-'proofreading for fidelity, but that doesn't usually cause any problem with internal mismatches in primers. We've found a more productive way of doing such mutations is to perform two independent PCRs with the mutation incorporated in both products. Those are then assembled into one longer product by crossover PCR (Pont-Kingdon, G. 1994. Biotechniques 16:1010), using only the primers at each far end of the assembled fragment. That is then inserted into plasmid by InFusion or Gibson Assembly. With relatively large constructs, and 9 Kbp is getting there, in our hands this has been a much more efficient approach in the long run. Good luck.

Hi,

I have sucess with the protocol which i am sending as attachment.There are certain drawback for using quickchange method which is clearly mentioned in this paper.However many people did get success with quick change also.The point i want to make is that you can try the protocol in the paper which uses non-overlapping primers.I have got success using the phusion enzyme and following the protocol in the paper.Hoping this will be helpful for you.

All the best.

Hi.

We very rarely see a product on a gel after performing the mutagenesis PCR because you have such a small amount of product. We just go straight to Dpn1 digestion and then transform into competent cells and usually get a good colony number with the plasmid successfully mutated - just don't forget to include a control without primers that you also Dpn1 digest and then transform (you shouldn't get any colonies from this).

Hope this helps!

@Frederick @Christian you guys were right! I did got four countable colonies and I got the DNA sequencing result also this week. Two of the clones were positive for the desired mutation. Thank you very much and thanks to the rest of the mates too for their suggestion!

@Shahid: Great news. And good luck for the further experiments.

Hi,

This may sound like heresy and it depends what you are trying to achieve (and how much money you have), but we have gone down the road of synthetic gene synthesis. You simply change the sequence as you want it and have it synthesised and sent to you in a plasmid ready to go and sub clone into your chosen vector. You can codon optimise for expression in your chosen organsism. The cost has really come down- £140 for mini genes (

@Noel: This might be true, if you have only one or two constructs, that need to be mutated. But if you want to test a number of different mutations on the same gene, all only be a few bases different, the PCR based site directed mutagenesis is cheaper and still very fast and straight forward. In terms of cloning sequences we also used this "synthetic" genes.

Hi Shahid,

Even though transforming cells without seeing bands in a gel can sometimes work, it is still a good control for the efficiency of your PCR reaction. I think you should lower your annealing temp to ~57C, and drop the elongation to no more than 10min. Also, do more cycles (25 - 30).

That's what I usually do, and most of the time - it works.

if you have a gradient function on your PCR machine, you can also do a temp gradient during your annealing step (52C - 62C). sometimes this works for harder mutations.

Finally, in the future, make sure your primers end with at least one C/G on both ends. this helps a lot.

Hi Shahid, From my experience though has not been much but yea SDM was n issue. Keep your samples in dpn fr long...I kept it for 16-18h once n tat too in incubator instead of water bath..lt is the PCR of the whole plasmid with mutations at some bsse.. so like a conventional PCR u wud not see definite bands but something kind of a smear. I got four transformed colonies n after seq found all four to b mutant...

Infact annealing temperature should be lower

Did you go ahead with transforming any of your SDM products? More often than not you might not see a band per se (since SDM reaction is not a typical PCR) but would still have sufficient amount of mutant template for transformation. Try transforming SDM product (DpnI treated) and you would be surprised. Good luck!

I would set lower annealing temperature (55ºC) and increases cycle number (you have a large plasmid)

Hi,

I want to synthesize active protein from non functional gene which is in human liver cells using site directed mutagenesis . Can you help me advising how I could that ?

In most cases, failed to get any PCR amplification product is due to the primer dimerisation, especially these designed based on the Quickchange protocol. Changing DNA polymerase hardly make any difference. It's better to redesign a new pair of primers and following the attached lab protocol summarized from the linked article http://www.biomedcentral.com/1472-6750/8/91

Hi Haroon,

When you do a site directed mutagenesis reaction you include very little amount of original plasmid in the reaction. A typical amount that I add is 0.1Ul from a plasmid solution of concentration 0.2mg/ml, thus only 20ng in 50 Ul reaction mix.  The idea is very simple; you add very little amount of plasmid, and introduce the mutations you want in the PCR products. Then you digest the original plasmid by adding DpnI into the reaction. Note that DpnI only digests the methylated DNA, the original plasmid is isolated  from E. coli source meaning that it is methylated. You will not digest your newly synthesized PCR products. Then you transform 2uL of this solution into a ultracompetent E. coli cells. You should get a lot of colonies even when you plate only 10Ul of the tranformation mix. I usually follow what the QuickChange guide suggests. So far it worked wonders for any mutation or deletion I made. I would refer to their handbook for more explanation.

A more direct answer to your question is that I have never tried running the PCR product on a gel. But I am guessing it would be very little amount, hence the use of ultracompetent cells ; )

Hope this helps! Good luck with your work.

Seyda

Dear Haroon,

mutating larger plasmids (>8 kb) with QickChange related methods is indeed more challenging. You might have a low concentration of the DNA construct. We therefore dialyze the sample after DpnI digestion prior to electrotransformation, to reduce the loss of DNA that occurs with purification columns. Make a control digest and transformation as well, so you are able to judge if the colonies might be undigested template. You might be lucky and get the mutant you aimed for as colony. As many have said here, an agarose gel might be a good control but not always are you able to see your band, since your nicked 8kb fragment might not be ideally linearized. Do you see a lot of material in your pockets? If so, go for transformation.

I would try phusion and increase the number of cycles to 25 or 30 instead of 20. Note that for phusion the efficiency of mutagenesis goes higher when using partially overlapping oligos (ideally 16-24 bases): https://www.ncbi.nlm.nih.gov/pubmed/25399421

Hello Shahid

Your query was also helpful for me.

Dear all,

I too have problem with SDM in 20kb BAC .

I am using Q5 SDM kit .

The plasmid preparation was done by in-house alkaline lysis method

(Sometimes this results in pellet with heavy salts even after ethanol precipitation).

I am interested in single substitution and the primers used were End to end aiming exponential increase in SDM PCR product.

The pregramme used for SDM was---

98-2min

30cycles of

98-30 sec

56 --58 : 45 sec

72: 20 min

Final extension 72-10 min

I was able to get a very faint band at 1 and 3 kb and nothing is visible at 20kb.

what might be affecting the yield of PCR product???

As shahid proceeded will it be wise to go for DpnI digestion and transformation????