Designing PCR Primers for cloning - (Apr/12/2013 )
Hello,
Urgently need some help on a question for an assignment that has had me stumped for 2 days.
I'm supposed to clone and express a gene that encodes a protein/enzyme known as CobT produced by the bacterium Mycobacterium avium paratuberculosis. Now I need to design a primer to help me amplify, clone and express the protein. Problem is I'm totally confused on how to go about this.
How do you design a primer? I know this is a super basic question but the multitude of tutorials I've seen have me all confused.
I tried using the ncbi primer blast tool but it seemed all the primers it gave me were inside my target sequence which makes no sense, how do you use the ncbi tool to pick primers?
Is there a difference between a primer for cloning and one for PCR?
Any help would be appreciated.
The CobT nucleotide sequence is attached below, the target sequence is highlighted in blue.
OK as you want the full coding sequence of the gene - you have no option about where you put the primers, which is why primer BLAST wasn't working for you. You need to use the first 20-25 bp of the gene and the reverse complement of the last 20-25 bp of the gene, including the start and stop codons respectively. Try and match the GC content and length to give similar annealing temperatures for the primers, but it isn't essential.
If you need to add restriction sites or anything else to the primers, these always go on the 5' end of the primer. If you are doing this you also need to add a few (usually 6) bp to the 5' end before the restriction site so that the RE has somewhere to bind.
Essentially there is no difference between primers for cloning and primers for ordinary PCR, other than that cloning primers frequently have tails added that contain restriction sites or tags (for the protein, e.g. flag tag) added. These aren't essential if you are doing TA cloning or blunt end cloning.
bob1 on Sat Apr 13 00:08:45 2013 said:
OK as you want the full coding sequence of the gene - you have no option about where you put the primers, which is why primer BLAST wasn't working for you. You need to use the first 20-25 bp of the gene and the reverse complement of the last 20-25 bp of the gene, including the start and stop codons respectively. Try and match the GC content and length to give similar annealing temperatures for the primers, but it isn't essential.
If you need to add restriction sites or anything else to the primers, these always go on the 5' end of the primer. If you are doing this you also need to add a few (usually 6) bp to the 5' end before the restriction site so that the RE has somewhere to bind.
Essentially there is no difference between primers for cloning and primers for ordinary PCR, other than that cloning primers frequently have tails added that contain restriction sites or tags (for the protein, e.g. flag tag) added. These aren't essential if you are doing TA cloning or blunt end cloning.
ATA TCA GAA AAA GCC AAA GCT GAT TGC AGT GCT TCT TCA GCG TAA TGC TCA GTT AAA AGC GAA GGT TGT
The primers look good to me. SOme people advocate putting a 3-6 bp between the restriction site and the start/stop codon, but it shouldn't be necessary.
taiju on Sat Apr 13 01:58:12 2013 said:
minor comments about your proposed primers:
the third triplet from the added bases of the reverse primer: you wrote "taa", i think you meant "tta"
you end both primers with a "t", it's good practice to end with a "g" or a "c" to clamp the primer.
mdfenko on Mon Apr 15 13:24:21 2013 said:
minor comments about your proposed primers:
the third triplet from the added bases of the reverse primer: you wrote "taa", i think you meant "tta"
you end both primers with a "t", it's good practice to end with a "g" or a "c" to clamp the primer.
Thanks, I hadn't spotted that.
About ending the primers with a "G" or "C"...the primers I made are basically just complements of my templates, to make them end with "G" or "C" is the idea to just shorten my primer until I get a "C" end? i.e for the forward primer:
FROM:
TO:
The recommended primer length of 18-30bp should include all these other bases right? So your (actual primer sequence+additional binding bases+restriction site and start and stop codons) should all add up to a maximum of 30?
If your sequence starts in ATG then its not really necessary to add a start codon right?
How would you use a primer design tool in such a way that it put your primers where you needed them, like in my case I need primers at the beginning of my coding sequence for the CobT protein as I want to be able to express CobT.
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Also concider this page
Cleavage Close to the End of DNA Fragments
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
taiju on Mon Apr 15 14:52:43 2013 said:
About ending the primers with a "G" or "C"...the primers I made are basically just complements of my templates, to make them end with "G" or "C" is the idea to just shorten my primer until I get a "C" end? i.e for the forward primer:
FROM:
TO:
The recommended primer length of 18-30bp should include all these other bases right? So your (actual primer sequence+additional binding bases+restriction site and start and stop codons) should all add up to a maximum of 30?
yes, sort of...
you can increase as well as decrease. also, you don't have to make the change with triads, you can change one base at a time so you could just remove the "tt" to get to the "g" or you could have added a "c" (or "cc") to the end to set up the clamp.
remember, you are extending from the end of the primer so you don't have to make changes in threes to maintain the coding sequence.
the necessary primer length takes into consideration only the actual primer sequence. the rest doesn't anneal.
mdfenko on Mon Apr 15 16:27:16 2013 said:
taiju on Mon Apr 15 14:52:43 2013 said:
About ending the primers with a "G" or "C"...the primers I made are basically just complements of my templates, to make them end with "G" or "C" is the idea to just shorten my primer until I get a "C" end? i.e for the forward primer:
FROM:
TO:
The recommended primer length of 18-30bp should include all these other bases right? So your (actual primer sequence+additional binding bases+restriction site and start and stop codons) should all add up to a maximum of 30?
yes, sort of...
you can increase as well as decrease. also, you don't have to make the change with triads, you can change one base at a time so you could just remove the "tt" to get to the "g" or you could have added a "c" (or "cc") to the end to set up the clamp.
remember, you are extending from the end of the primer so you don't have to make changes in threes to maintain the coding sequence.
the necessary primer length takes into consideration only the actual primer sequence. the rest doesn't anneal.
You just answered a question I was about to ask, thank you as I was wondering about what happens to the rest of the bases you add but if they don't anneal how do you get an amplicon that has the restriction site and the start/stop codons, like if only the actual primer sequence anneals and is replicated and the additional bases are just kind of hanging how do they get into the subsequently produced PCR products.. I hope my question makes sense.
The primer ends get copied by the subsequent cycles of PCR, its just the initial cycles where there are "free" ends.