Cloning large gene - (Jul/30/2011 )
Hi
I am trying to clone cDNA for a gene where the mRNA is 14kb. I have managed to PCR out the gene in 3 segments (5' 4kb-5Kb-5Kb 3') with unique restriction sites so that I can stitch the 3 segments together into an expression vector to get the full intact cDNA, two of the restriction sites are found naturally within the gene (AfeI and SexAI) and I introduced two myself, one at either end of the gene to allow me to put the cDNA into pCi. I TOPO cloned the segments and produced a multiple cloning cassette in pCi containing the restriction sites 5' KpnI, AfeI, SexAI and SalI 3' to allow me to stitch up the different fragments in the correct order.
I have managed to add on the 3' fragment using the SexAI and SalI restriction sites but I when I try to add the AfeI-SexAI segment all my colonies are false positives, I am using fastdigest restriction enzymes just so that I could use the same buffer for the different enzymes, I always leave the enzymes to digest for at least an hour, have also tried overnight but it doesn't help. I am transforming the digestion product using electroporation (using heat-shock gives no colonies which itself is weird as when I added the 3' fragment I used heat-shock to transform and got plenty of colonies and a high efficiency of positive colonies so surely the false positives should show up when using heat-shock??). I have also tried using CIP and get a reduction in colonies but they are all still false positives.
Does anyone have any ideas as to what could be going wrong or any different approaches I could try?
It's probably because those natural RE sites are not there anymore because of mutation or because the sequences that you have are not very reliable after all. the other RE sites that you introduced by yourself work fine, meaning there is no problem with your Fermentas fast-digest system.
Interestingly I am doing the same thing on a 15 kb virus genome. I have 8 fragments however. But I tend to sequence the genome by myself, before designing the primers. In our case, viruses mutate very fast, and after one propagation the next batch of virus might not have the same sequence.
Therefore, it would have been better for you to sequence that region again and design other primers. Do you understand what I'm saying? good luck
also, did you use pfu polymerase? probably the AfeI is not there.
Curtis on Mon Aug 1 18:46:47 2011 said:
It's probably because those natural RE sites are not there anymore because of mutation or because the sequences that you have are not very reliable after all. the other RE sites that you introduced by yourself work fine, meaning there is no problem with your Fermentas fast-digest system.
Interestingly I am doing the same thing on a 15 kb virus genome. I have 8 fragments however. But I tend to sequence the genome by myself, before designing the primers. In our case, viruses mutate very fast, and after one propagation the next batch of virus might not have the same sequence.
Therefore, it would have been better for you to sequence that region again and design other primers. Do you understand what I'm saying? good luck
also, did you use pfu polymerase? probably the AfeI is not there.
No it doesn't really make sense, I am confused as to what you think I am trying to do. I have already cloned the fragments into TOPO vector and have them sequenced, I have also been able to cut out the fragments from the TOPO vector fine using the enzymes for the natural RE sites i.e. AfeI and SexAI. I am struggling to ligate the fragments into a pCi vector to get the full intact cDNA in one place. With the pCi vector I have introduced a multiple cloning cassette with all the unique restriction sites KpnI, AfeI, SexAI and SalI as mentioned above and have managed to ligate in the 3' segment of my gene of interest (flanked by SexAI and SalI) but can't get any of the other segments in, I just get lots of false positives.
Mustafa on Tue Aug 2 21:57:51 2011 said:
Curtis on Mon Aug 1 18:46:47 2011 said:
It's probably because those natural RE sites are not there anymore because of mutation or because the sequences that you have are not very reliable after all. the other RE sites that you introduced by yourself work fine, meaning there is no problem with your Fermentas fast-digest system.
Interestingly I am doing the same thing on a 15 kb virus genome. I have 8 fragments however. But I tend to sequence the genome by myself, before designing the primers. In our case, viruses mutate very fast, and after one propagation the next batch of virus might not have the same sequence.
Therefore, it would have been better for you to sequence that region again and design other primers. Do you understand what I'm saying? good luck
also, did you use pfu polymerase? probably the AfeI is not there.
No it doesn't really make sense, I am confused as to what you think I am trying to do. I have already cloned the fragments into TOPO vector and have them sequenced, I have also been able to cut out the fragments from the TOPO vector fine using the enzymes for the natural RE sites i.e. AfeI and SexAI. I am struggling to ligate the fragments into a pCi vector to get the full intact cDNA in one place. With the pCi vector I have introduced a multiple cloning cassette with all the unique restriction sites KpnI, AfeI, SexAI and SalI as mentioned above and have managed to ligate in the 3' segment of my gene of interest (flanked by SexAI and SalI) but can't get any of the other segments in, I just get lots of false positives.
Oh and I also have a 10bp between each restriction sequence site which I am told is more then enough, I have also sequenced the pCi vector and confirmed the introduced restriction sites are there and also cut the vector with all the restriction enzymes individually, in all 4 cases the restriction enzymes linearized the plasmid so I know the restriction sequences are there.