Hello
I have been trying to make deletion constructs using site directed mutagenesis. The largest deletion is around 522 basepairs. My plasmid is around 11KB. I have tried inverse PCR primers, complementary primers, and primers that are only complementary at the 5' end with 3'end overhangs. I have used the Agilent kits, NEB kits and have tried it manually using different master mixes. I have tried varying extension times and played around with the DMSO concentrations. But nothing seems to work and I mostly get one or two colonies of parental plasmids or incomplete sequences with no deletions. I would be extremely grateful, if anyone could give me a few tips..
Thank you
Pranetha
DEar Praneta
did you check the PCR on the gel?
Because 11Kb, is quite big plasmid and not al the Taqs are able to amplify so long constructs
However i suggest to you to try the PIPE cloning (Article Combining the polymerase incomplete primer extension method ...
)For a so long PCR i suggest to you to screen more polimerase: Kapa Hif (Roche)i, Clona amp HIfi (Takara) or Pfu ultra Fusion II (Agilent) are 3 possible candidates. In my experience, it work all well p to 8-9 Kb but sincerellyi never tried on a so long construct.
If you would like to know some more details about primer desing for PIPE cloning you can find a presentation named ProteoCool n° 3:Primer desing for PIPE Cloning
on my blog: http://proteocool.blogspot.com/
i wish that it can help you in some way.
good luck
Manuele
Hi
Potentially another method makes it easier. I would suggest to use overlap extension PCR as this is reducing the amplicon size and increases reaction robustness. I can check for the protocol if needed
Sven
Dear sir/madam
One thing
Can be reduce the dmso concentration
Little mg++ can be added
Hi have you tried usin phusion reaction for your mutagenesis experiment. If you have done inverse PCR, I am interested in the concentration of your template used in the reaction . I assume that though you get colonies but it happens to be parental plasmid this could be due to high concentarion of template used in your PCR reaction. For deletions it is easier to use NEB primer design tool, it helps a lot. Also consider Dpn I digestion after your PCR, phoshoralating your product before ligation but if your primers are phosphorylated this step is not essential.
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