restriction digestion - (May/29/2007 )
Hi all
I am cloning a 900 bp fragment in a binary vector. I get positive result for colony pcr with the primers of the gene but I'm unable to release the stuff out of the vector using the sites near by the insert. I'm using BamHI and HindIII to release the insert. Can anyone help me? Thanks in advance
There are a couple potential problems here...pcr of a colony can give false positive results as some of the insert may be just sitting on the plate and you happen to pick up just a bit of it with the bacteria. This is rare but I've had it happen to me. Otherwise, how are you doing the digest? According to NEB you must do a BamHI and HindIII digestion sequentially. If you are just attempting the double digest you will not get your insert out. Hope this helps a bit.
Thank you sir.
I got 15 colonies and all this seems to be positive by colony pcr. And I dont understand how you say I could have picked the insert from plate.
As you have said I am doing double digestion bcs the company enzyme i use says 100% activity for both the enzymes in a buffer. Anyway I will try sequential digestion.
Thank you sir.
I got 15 colonies and all this seems to be positive by colony pcr. And I dont understand how you say I could have picked the insert from plate.
As you have said I am doing double digestion bcs the company enzyme i use says 100% activity for both the enzymes in a buffer. Anyway I will try sequential digestion.
Hello,
What primers you used in your colony PCR? the forward and reverse primers are gene specific??? or one is gene specific and another is vector primer???? If u use the gene specific primers(forward and reverse), you may get the false positive results.
Did you check the size of the digested plasmid? According to size of the digested plasmid, u may have idea whether the plasmid contains your insert even it was cut by one enzyme only.
Hope this can help.
what is the result of a pcr in a "negative" colony PCR ? you may amplify something irrelevant in your pcr conditions
if you're convinced you get smething interesting, sequence it.
Thanks for all your suggestions. I have used gene specific primer both fwd and rev. I get the expected size of amplicon in colony pcr along with that I do get three extra bands too..... I have maintained positive control to compare my amplicon. The negative control shows some bands but irrelevant... These results seems to convince my prof for a positive result because of the amplicon size. He wants me to proceed to southern blotting.