Site-directed mutagenesis - (May/13/2012 )
One of my friends said that I should use
Anyone who has better ideas please tell me!
Thank you!
I needed to do a SDM on 12K vector a while back. I didn't have the Stragene kit and decided to give our high fidelity polymerase (AccuPrime Pfu, Invitrogen) a try. I designed the primers with the Strategen web tool and ordered the primers. Since the primers were long, I asked the company to purify them on PAGE (this takes a longer time and more money, so it's not going to be a 10$ per pair primer). Then I borrowed the dpnI from the neighbors and that did it! all my colonies were mutant. Here is my thoughts:
1) Are you sure about your primer design? They have to be overlapping and long.
2) Did you purify the primers?
3) Do you give the polymerase enough time? (12 minutes should be fine, but I would give it a little longer)
4) I might be wrong, but a longer dpnI treatment might help to eliminate more of the original plasmid.
5) How do you check for the mutation? sequencing?
kaveh on Mon May 14 04:45:05 2012 said:
I needed to do a SDM on 12K vector a while back. I didn't have the Stragene kit and decided to give our high fidelity polymerase (AccuPrime Pfu, Invitrogen) a try. I designed the primers with the Strategen web tool and ordered the primers. Since the primers were long, I asked the company to purify them on PAGE (this takes a longer time and more money, so it's not going to be a 10$ per pair primer). Then I borrowed the dpnI from the neighbors and that did it! all my colonies were mutant. Here is my thoughts:
1) Are you sure about your primer design? They have to be overlapping and long.
2) Did you purify the primers?
3) Do you give the polymerase enough time? (12 minutes should be fine, but I would give it a little longer)
4) I might be wrong, but a longer dpnI treatment might help to eliminate more of the original plasmid.
5) How do you check for the mutation? sequencing?
Hi
(1)My primer design is from
i have done lot of SDMs and i never had failure untill now...your protocol looks ok, but your PCR cycle shdnt be 30. may be 15 shd be more than enough. after dpn digestion do a gel purification and transform. my usual primer design tool is Primer X.....i use Accuprime pfx from invitrogen.....
good luck...
GNANA on Mon May 14 09:05:58 2012 said:
i have done lot of SDMs and i never had failure untill now...your protocol looks ok, but your PCR cycle shdnt be 30. may be 15 shd be more than enough. after dpn digestion do a gel purification and transform. my usual primer design tool is Primer X.....i use Accuprime pfx from invitrogen.....
good luck...
Hello Veteran:
I've done 18cycles before, but I got no colonies.
Thanks for advises!
Did you check on gel after PCR done with 18 cycles??. bcoz no colonies might also be due to lack of adequate competency in your competent cells. anyways doing 30 cycles on 12 kb vector is not recommended as your enzyme availability and fidelity for the entire 30 cycles is a matter of concern. even i you get positive SDMs there is also possibility of other mutations with your 30 cycle PCR. so i would suggest you to standardize your PCR to get a band in 15 cycles or around.
GNANA on Tue May 15 08:33:07 2012 said:
Did you check on gel after PCR done with 18 cycles??. bcoz no colonies might also be due to lack of adequate competency in your competent cells. anyways doing 30 cycles on 12 kb vector is not recommended as your enzyme availability and fidelity for the entire 30 cycles is a matter of concern. even i you get positive SDMs there is also possibility of other mutations with your 30 cycle PCR. so i would suggest you to standardize your PCR to get a band in 15 cycles or around.
Hi
Reg your competent cells taking up the plasmid doesnt mean it is enough competent to take up and process the nicked , and relaxed plasmid produced by the Linear amplification. so make your the efficiency of your competent cells if it is home made.
getting original plasmid suggests your Dpn digestion is not complete, so have a check over there as well.
Theoritically its true (strategenes guidelines) , but i personally dnt believe unless i see....anyways how you made sure your PCR worked??? did you see band with 30 cycle PCR?? how much PCR you run to check the band?? if it s just below 5% of total volume you may not see but i would suggest, after dpn digestion run the whole PCR and look for band, here you definitely shd see the band if your PCR worked, eventually you can do a gel purification and transformation of whole product or part of it depending on the band intensity....if not you still got to standardize your PCR rather than troubleshooting downstream.
good luck,
Gnana...
If you're getting that many mutant colonies, then either something is wrong with your miniprep (dirty or denatured DNA that resists DpnI digest) or your primers are binding in the wrong place. Try alternative primer design strategies, such as one described by Liu and Naismith, BMC Biotech 8:91.
GNANA on Wed May 16 08:22:58 2012 said:
Reg your competent cells taking up the plasmid doesnt mean it is enough competent to take up and process the nicked , and relaxed plasmid produced by the Linear amplification. so make your the efficiency of your competent cells if it is home made.
getting original plasmid suggests your Dpn digestion is not complete, so have a check over there as well.
Theoritically its true (strategenes guidelines) , but i personally dnt believe unless i see....anyways how you made sure your PCR worked??? did you see band with 30 cycle PCR?? how much PCR you run to check the band?? if it s just below 5% of total volume you may not see but i would suggest, after dpn digestion run the whole PCR and look for band, here you definitely shd see the band if your PCR worked, eventually you can do a gel purification and transformation of whole product or part of it depending on the band intensity....if not you still got to standardize your PCR rather than troubleshooting downstream.
good luck,
Gnana...
I load 1/3 of my PCR product. Actually I am not sure my PCR works or not. I hope it's work! But I can't see band.
Thank you~