Mutagenesis not working - no colonies (Oct/23/2006 )
Hey all,
I want to introduce a single amino acid change in my template to transfect cells afterwards, however I get no colonies.
In the first attempt, I used the Quick Change II Site Directed Mutagenesis Kit from Stratagene. But one of my colleagues used up the Pfu Polymerase and the Enzyme which comes with the kit , so I used bought Pfu polymerase and DpnI from Fermentas but with the same concentrations used in the kit.
Here's what I've done:
5 µl Reaction buffer (10x)
1 µl Primer F (125ng)
1 µl Primer R (125ng)
1 µl dNTP Mix
1 µl DNA (10ng)
41 µl water
+ 1µl Pfu polymerase
PCR cond:
95 °C 30s
95 °C 30s
55 °C 1min
68 °C 6min (1min/kb) 16 cycles
68 °C 10min
4 °C cooling
I got the expected results from the PCR, checked on a 1% agarose gel. Digested the parental strand with DpnI for one hour, didn't check the digest though, transformed 1µl of the remaining 40µl - no colonies!!! Transformed 5 µl of the digest - no colonies. I used XL-1 comp. cells which were made in our lab, they are relatively fresh and have worked every time I used them before.
Then I've transformed 1 µl into commercial Top Ten Cells from Invitrogen - no colonies
Ok, now I've tried a recipe someone posted here:
5 µl Reaction buffer (10x)
1 µl Primer F (125ng)
1 µl Primer R (125ng)
1 µl dNTP Mix
1 µl DNA (50ng)
41 µl water
+ 1µl Pfu polymerase (2.5U)
PCR cond:
95 °C 30s
95 °C 30s
55 °C 1min
68 °C 12min (2min/kb) 16 cycles
68 °C 20min
4 °C cooling
I will check on a gel again, check also the digest and try to transform into XL-1 cells. But if anyone can see what I'm doing wrong or what I should try, please don't hesitate. It's really frustrating!!!
Hi there!
I'm not familiar with your kit, but I 'v e done some SDM myself with a homegrown system. And maybe I'm missing something, but in my case I had to restriction digest the PCR product and re-ligate it. Without ligase, not circular plasimd, only linear PCR product, which can't replicate in E.coli.....
And in my experience is a 1h DpnI digest more than enough to digest parental DNA (coming from a E.coli starin that's comatible to a DnpI digest), I usually only incubate it for 30', and when checked, there's nothing there anymore....So that should not pose a problem.
Maybe this helps.
Mike
I already have my target DNA in pEYFP, so it should work shouldn't it??? Maybe you can explain your attempt just a little bit more, I'm thankful for any info you've got.
you have to use a particular E coli strain that repair the plasmid (there is no ligation)
I can't find the name right now, maybe later.
I can't find the name right now, maybe later.
I got it : the epicurian E coli.
they correct the plasmid, but the yield is lower than an other bacteria, so I suggest you to make a miniprep from epicurian bacteria, and then transform an other coli strain.
I am so sorry, I don't get it.
And if I have to do this repairing stuff, how come Stratagene doesn't tell me???
It seems nothing wrong ...
Is 12 min enough for your template? I did 12.5min for my vector.. Did you check the plate? sometimes the selection has problem.. Did you do a positive control, or you used this protocol successfully before?
And if I have to do this repairing stuff, how come Stratagene doesn't tell me???
when you amplify your plasmid, there is a gap between the beginning of your primer, and the end of the polyerised plasmid, because you don't use a ligase.
You get a double stranded plasmid where the oligonucleotide before the 5' of the primer is not linkded to the 5', on each strand.
the epicurian coli XL1-blue that is provided in the kit (?) is able to repair this little hole.
I don"t know if your XL1 blue are the same than the epicurian one.
so in fact you don't have to repair, the bacteria does it for you.
@missele: you might have a point there, however, Stratagene does not tell me if the provided cells (which also seem to have vanished from the freezer, thanks to whoever used them before) are these epicurian ones.
Stratagene only tells me this: Genotype: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´ proAB lacIqZΔM15 Tn10 (Tetr)]
I'm sorry, I can't tell you if this genotype is the right one.
Try to find someone in the institute that is doing quickchange successfully and ask for the bacteria, or contact strategen.