PCR using long primers and two overlapping templates - PCR using long primers and two overlapping templates (Jun/02/2006 )
Hello,
I need to create the following fragment for subsequent ligation into a lentiviral vector GFP-Peptide2A-Gene of Interest. This will enable me to obtain a bicistronic lentiviral expression vector as with an IRES the longer ORF's I have to clone resulted in substantially reduced expression of both my gene of interest and GFP. I have two queries:
1. To do this I was planning to amplify GFP using a 'normal' forward primer and the reverse primer would be the 3' region of the GFP sequence and also the 69 nucelotides of the Peptide 2A sequence. This seems excessively long for a primer but should it work or does anyone have better ideas to overcome this?
2. The second part was to amplify the gene of interest with a 5' overlapping seqeunce to the Peptide 2A sequence. Then I would perform a third PCR using the products from the initial two PCRs. Do these two products need to be gel purified before performing the PCR or would a simple PCR clean up suffice.
Thanks in advance
does anyone have any suggestions?
I need to create the following fragment for subsequent ligation into a lentiviral vector GFP-Peptide2A-Gene of Interest. This will enable me to obtain a bicistronic lentiviral expression vector as with an IRES the longer ORF's I have to clone resulted in substantially reduced expression of both my gene of interest and GFP. I have two queries:
1. To do this I was planning to amplify GFP using a 'normal' forward primer and the reverse primer would be the 3' region of the GFP sequence and also the 69 nucelotides of the Peptide 2A sequence. This seems excessively long for a primer but should it work or does anyone have better ideas to overcome this?
2. The second part was to amplify the gene of interest with a 5' overlapping seqeunce to the Peptide 2A sequence. Then I would perform a third PCR using the products from the initial two PCRs. Do these two products need to be gel purified before performing the PCR or would a simple PCR clean up suffice.
Thanks in advance
This previous post may be interesting for you:
"Join GFP to my insert without mutagenesis"
http://www.protocol-online.org/forums/inde...&hl=mutagenesis
I haven't got any successful result from the fusion PCR, but I'm hoping I will achive it soon.
About the second question, in my view a PCR clean up should be OK (don't forget to check that you only get a clear band in a gel using a 5ul from the PCR product). However, I prefer to do gel purification in all cases.