I have performed Quick-change site directed mutagenesis by using stratagene protocol (Not kit). Mutagenesis primers were exact complementary and I have performed
1.Independent F & R primer
2. simultaneous FR PCR 10ng template in 25ul reaction and 20ng template DNA in 50ul reaction.
After PCR and before DpnI digestion:-
I run gel for (simultaneous PCR) 50ul reaction (10ul PCR product loaded on gel) and getting expected size single band.
After DpnI digestion of 1 hr (without heat inactivation):-
I run gel for both 25ul and 50 ul simultaneous PCR DpnI cut samples, here I got band only for 50ul reaction, position and size of this band was similar as after PCR amplification gel. What is the meaning of similarity of PCR amplificaion and DpnI digestion band for 50 ul reaction?
Absence of band for 25ul reaction after DpnI digestion what does that mean?
For all sample I got sufficient number of colonies after transformation.
On the basis of gel results, Is mutagenesis working or not ?
This was my second attempt as there was no luck during first attempt.
Hello Monika,
I am not sure I understand everything in your description. However :
1. The major species that you should see after the PCR reaction is the mutated species; it should be resistant to DpnI since it is not methylated
2. The non mutated, methylated template is still present after the PCR reaction; although it is much less abundant than the mutated PCR product, it will transform much more efficiently, hence it is important to destroy it with DpnI. But since it is present in very low amount, it may escape detection on the gel even before the treatment with DpnI. Thus, the gel pattern looks the same before and after DpnI, since the only species detected is the mutagenized, DpnI resistant species
3. Most important, if you got colonies from the DpnI-treated sample, these are most likely mutants, so just pick a few them, make plasmid minipreps and have them sequenced to verify that the mutation is there and that there are no further, spurious mutations that could have been introduced by the PCR reaction.
Good luck,
Piere
Hello Monika,
I am not sure I understand everything in your description. However :
1. The major species that you should see after the PCR reaction is the mutated species; it should be resistant to DpnI since it is not methylated
2. The non mutated, methylated template is still present after the PCR reaction; although it is much less abundant than the mutated PCR product, it will transform much more efficiently, hence it is important to destroy it with DpnI. But since it is present in very low amount, it may escape detection on the gel even before the treatment with DpnI. Thus, the gel pattern looks the same before and after DpnI, since the only species detected is the mutagenized, DpnI resistant species
3. Most important, if you got colonies from the DpnI-treated sample, these are most likely mutants, so just pick a few them, make plasmid minipreps and have them sequenced to verify that the mutation is there and that there are no further, spurious mutations that could have been introduced by the PCR reaction.
Good luck,
Piere
You got colonies after DpnI digestion...!! go for colony PCR, sequencing to identify mutant colonies than wasting time on gel results. Note: Mutant plasmids and wild type plasmids wont show a difference in gel. Good luck..!!
Hello Jinesh..
Yes I got colonies after Dpn I digestion.
How colony PCR will differentiate mutant and wild type colonies ?
During my first trial there was no luck in sequencing result, I have repeated it , now hoping for positive result.
Colony PCR will amplify the area to be sequenced, cut+elute the PCR product using a GFX column or qiagen gel purification column and subject the purified DNA for sequencing. Othe way is to find restriction sites specific for mutants but the cost of restriction enzymes for screening will be too much compared to sequencing these days. Since its a PCR from plasmids, the sequencing should work with cleaner peaks. Make sure you save remaining colony for future use after sequencing confirmation.
As Jinesh said , sequencing is the most fast and reliable method to confirm that your mutagenesis works.
Actually i never relay on the gel results before or after DpnI digestion beacuse i did alot of mutagenesis using stratgene protcol and sometimes i didn't see bands in the gel and when i transform the competent Ecoli , i can get a colonies with my mutants , this beacuse that the mutagensis PCR reaction is not exponential like normal PCR.
I would like you to take care of 2 major issues while doing your mutagenesis
1- The concentration of template plasmid in your mutagenesis reaction, if you used high conc. of plasmid DNA (Template) , this might inhibit your reaction , and later on when you proceed with the DpnI digestion , some of your WT plasmid will be till there and give you colonies after transformation beacuse the DpnI couldn't digest all of your WT plasmid (due to high template conc.)
2- I recommend you to perform longer incubation for DpnI digestion , 2 hours , this much better to be sure that the DpnI digested all of your WT plasmid.
Wish you all luck.
Regards
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