Cloning PCR product blunt + restriction site - (Oct/09/2007 )
Hi there,
I'm Peter and I'm starting a research project in which I'd like to clone PCR products into the New England BioLabs pMAL protein fusion vector. In this vector, the gene to be expressed is ligated into the vector downstream of an N-terminal tag sequence.
One of the strategies suggested in the manual is to create a PCR product using primers that give a 5' blunt end and a 3' end with a restriction site created by the reverse primer. In this strategy, in the PCR the forward primer will anneal exactly to the target gene, while the reverse primer has a 5' overhang in the initial PCR cycles. After a few cycles, both primers will anneal completely and perfectly to the target.
My first question:
Should I design my PCR primers in such a way that ....
a. the Tm of the forward primer matches the Tm of the complete reverse primer (including the 5' tail containing the restriction site)? OR
b. the Tm of the forward primer matches the Tm of only the part of the reverse primer that matches the target sequence (without the 5' tail containing the restriction site)?
My second question:
I saw in the famous table form New England BioLabs that EcoRI only needs one extra bp in the PCR product to still efficiently cut the end of the PCR product. In other words: when adding an EcoRI site to the downstream primer the addition of one extra nucleotide 5' to the restriction site would suffice. Does anyone have experience in using EcoRI in this way? Is one extra nucleotide really enough? That would be nice, since I want to keep the addition of extra nucleotides to the downtream primer to a minimum (because I'm using an upstream primer WITHOUT extra nucleotides).
My third question:
I have found a region downstream of the ORF of the gene I want to clone that resembles an EcoRI site. I've designed a mutagenic downstream primer (with two mismatches in the middle of the primer) that introduces an EcoRI site into the PCR product. Any suggestions concerning this approach (Tm of the upstream primer, annealing temperature to use in the PCR etc.) would be highly appreciated!
Thanx very much!
Peter
I'm Peter and I'm starting a research project in which I'd like to clone PCR products into the New England BioLabs pMAL protein fusion vector. In this vector, the gene to be expressed is ligated into the vector downstream of an N-terminal tag sequence.
One of the strategies suggested in the manual is to create a PCR product using primers that give a 5' blunt end and a 3' end with a restriction site created by the reverse primer. In this strategy, in the PCR the forward primer will anneal exactly to the target gene, while the reverse primer has a 5' overhang in the initial PCR cycles. After a few cycles, both primers will anneal completely and perfectly to the target.
My first question:
Should I design my PCR primers in such a way that ....
a. the Tm of the forward primer matches the Tm of the complete reverse primer (including the 5' tail containing the restriction site)? OR
b. the Tm of the forward primer matches the Tm of only the part of the reverse primer that matches the target sequence (without the 5' tail containing the restriction site)?
My second question:
I saw in the famous table form New England BioLabs that EcoRI only needs one extra bp in the PCR product to still efficiently cut the end of the PCR product. In other words: when adding an EcoRI site to the downstream primer the addition of one extra nucleotide 5' to the restriction site would suffice. Does anyone have experience in using EcoRI in this way? Is one extra nucleotide really enough? That would be nice, since I want to keep the addition of extra nucleotides to the downtream primer to a minimum (because I'm using an upstream primer WITHOUT extra nucleotides).
My third question:
I have found a region downstream of the ORF of the gene I want to clone that resembles an EcoRI site. I've designed a mutagenic downstream primer (with two mismatches in the middle of the primer) that introduces an EcoRI site into the PCR product. Any suggestions concerning this approach (Tm of the upstream primer, annealing temperature to use in the PCR etc.) would be highly appreciated!
Thanx very much!
Peter
I would design primers such that I have the sites on both ends and then to digest them even if its a blunt end or sticky end. This way you can be sure the ends are the way you wanted them to be. I always leave 5-6 bases after the restriction sites to allow for proper digestion.
Design primers such that there is atleast 21 bases overlap and don't worry too much about the Tm.
I'm Peter and I'm starting a research project in which I'd like to clone PCR products into the New England BioLabs pMAL protein fusion vector. In this vector, the gene to be expressed is ligated into the vector downstream of an N-terminal tag sequence.
One of the strategies suggested in the manual is to create a PCR product using primers that give a 5' blunt end and a 3' end with a restriction site created by the reverse primer. In this strategy, in the PCR the forward primer will anneal exactly to the target gene, while the reverse primer has a 5' overhang in the initial PCR cycles. After a few cycles, both primers will anneal completely and perfectly to the target.
My first question:
Should I design my PCR primers in such a way that ....
a. the Tm of the forward primer matches the Tm of the complete reverse primer (including the 5' tail containing the restriction site)? OR
b. the Tm of the forward primer matches the Tm of only the part of the reverse primer that matches the target sequence (without the 5' tail containing the restriction site)?
My answer to this question is b. Only the segment of the primer that binds to the template is actually considered in the annealing temperature calculation.
I saw in the famous table form New England BioLabs that EcoRI only needs one extra bp in the PCR product to still efficiently cut the end of the PCR product. In other words: when adding an EcoRI site to the downstream primer the addition of one extra nucleotide 5' to the restriction site would suffice. Does anyone have experience in using EcoRI in this way? Is one extra nucleotide really enough? That would be nice, since I want to keep the addition of extra nucleotides to the downtream primer to a minimum (because I'm using an upstream primer WITHOUT extra nucleotides).
I have never tried 1bp. The shortest I have tried is 4bp. However I don't seem to understand the question. Let say your primers was as below. I don't see why down steam
NNNNN GAATTC NNNNNNNNNNNNNN
NNNNN forms the guard sequence that buffers the EcoRI restriction site.
NNNNNNNNNNNNNN which is the template binding sequence.
On digestion, the entire guard sequence is removed. No extra sequence aside from the EcoRI site will be incoperated into the final ligated product
Perhaps interperated another way; if you are trying to save bp and make short (cheaper) primer- well 100bp is around the limit Invitrogen will sell you. No additional features have been added to your primer, so with only a template binding sequence and a restriction site, there should be plenty of space left. 4bp isn't going to save you much in the long run.
I have found a region downstream of the ORF of the gene I want to clone that resembles an EcoRI site. I've designed a mutagenic downstream primer (with two mismatches in the middle of the primer) that introduces an EcoRI site into the PCR product. Any suggestions concerning this approach (Tm of the upstream primer, annealing temperature to use in the PCR etc.) would be highly appreciated!
Well be careful, don't forget genes need terminator sequences as well. Either you add one yourself to your gene's ORF or amplify enough 3' region to hopefully capture said region. As for Tm... well like any thing, calculation can predict, but the only real way to find out is to do the experiment. That is why there is much fun and games with PCR optimisation. My favourite Tm is 60 Celsius. It makes it easier for primers from different projects to work together (or in the same heating block and saves time) as their tm are near identical.