Directional cloning and clone mutations - each clone has a different mutation! (Aug/31/2005 )
Hi
I cloned a 1.4kb fragment of a promoter into pGL3 Basic (a Promega promoterless LUC expression vector).
This is how I did it the first time:
1. PCR-amplified promoter region from genomic mouse DNA using LA Taq.
2. Re-amplified off gel slice using (nested) primers with restriction sites, again using LA Taq.
3. Cut PCR and vector with rest. enzymes, gel purified and put into ligation, transformed.
4. Yay! Clones!!!....or so I thought.
I sequenced my clones, comparing it to the sequence I obtained from genomic DNA. Every single clone (about 10 or so) had a mutation or two in it, somewhere different from the others. I thought my second amplification using the nested primers and LA Taq was suspect (maybe introducing my mutations), so I repeated the process, this time using Ex Taq with the nested PCR. I just checked one of my clones, and lo and behold, I have mutations again!!!
So what the heck is going on here? Is it possible my DH5alphas are at fault here? I'm scared to even check the sequence of my other clones for fear of more mutations. Can anyone tell me if this has happened to you before? I don't understand how clones from the same transformation can all be different in sequence.
Oh, by the way, I also followed the first procedure with a different mouse line, and was able to get an error free clone out of it ( I did it at the same time as the other one).
I am really lost here and getting frustrated. Any help would be much appreciated.
~Sheila
Hi Sheila,
Taq is not the best enzyme to use in PCRs you want to clone. It lacks proofreading ability (ie when it makes mistakes it doesn't realise it).
I strongly suggest you use a polymerase that has this ability, like Pfu.
You could try to avoid the second PCR step by doing an intermediate TOPO cloning and using the cutting sites in the multiple cloning site of the TOPO-vector to put your sequence into your vector.
Alternatively you could use Taq in the first PCR (as few cycles as possible - start with more template and ul) and do something called TA cloning.
I cloned a 1.4kb fragment of a promoter into pGL3 Basic (a Promega promoterless LUC expression vector).
This is how I did it the first time:
1. PCR-amplified promoter region from genomic mouse DNA using LA Taq.
2. Re-amplified off gel slice using (nested) primers with restriction sites, again using LA Taq.
3. Cut PCR and vector with rest. enzymes, gel purified and put into ligation, transformed.
4. Yay! Clones!!!....or so I thought.
I sequenced my clones, comparing it to the sequence I obtained from genomic DNA. Every single clone (about 10 or so) had a mutation or two in it, somewhere different from the others. I thought my second amplification using the nested primers and LA Taq was suspect (maybe introducing my mutations), so I repeated the process, this time using Ex Taq with the nested PCR. I just checked one of my clones, and lo and behold, I have mutations again!!!
So what the heck is going on here? Is it possible my DH5alphas are at fault here? I'm scared to even check the sequence of my other clones for fear of more mutations. Can anyone tell me if this has happened to you before? I don't understand how clones from the same transformation can all be different in sequence.
Oh, by the way, I also followed the first procedure with a different mouse line, and was able to get an error free clone out of it ( I did it at the same time as the other one).
I am really lost here and getting frustrated. Any help would be much appreciated.
~Sheila