I want to include loxP sites flanking a gene that I want to amplify and then clone it into an expression vector. This is around 34 bp (+ 6 bp for a restriction site) that I must tag on to my primers. Any feedback will be appreciated.
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What's the longest overhanging primer seq. you have used?
Started by snpsnooper, Jun 18 2010 09:16 AM
2 replies to this topic
#2
pDNA
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Posted 18 June 2010 - 11:02 AM
should be absolutely no problem ...use 50bp overhangs without any problems regularly.
Regards,
p
Regards,
p
#3
Curtis
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Posted 19 June 2010 - 09:37 AM
6 mer is quite ok, I've gone up to 8 for overhangs.
A friend of mine used 42 mer primer last time and she had no problem.
in case your PCR product digestion doesn't work, first clone the PCR product into a TA vector, or CloneJet vectors from Fermentas, then digest and clone into the second vector. This way you'll end up with many more colonies.
A friend of mine used 42 mer primer last time and she had no problem.
in case your PCR product digestion doesn't work, first clone the PCR product into a TA vector, or CloneJet vectors from Fermentas, then digest and clone into the second vector. This way you'll end up with many more colonies.
snpsnooper, on Jun 18 2010, 09:16 AM, said:
I want to include loxP sites flanking a gene that I want to amplify and then clone it into an expression vector. This is around 34 bp (+ 6 bp for a restriction site) that I must tag on to my primers. Any feedback will be appreciated.
