cloning issues - (Sep/21/2014 )

Hi Guys-
 
I'm a cloner-in training! and wanted to check with all of you if I have my PCR will work with this primer set to amplify a full length cDNA.
 
Forward primer:
 ACC GGT GCC ACC ATG GAT TAC AAG GAT GAC GAT GAC AAG CCC CTC AAC GTT AGC TTC A

      1                2                                      3                                                               4

1) restriction site, 2) Kozak, 3) FLAG tag including ATG, 4) gene sequence after ATG for my cDNA

Reverse Primer:

ACG CGT TTA CGC ACA AGA GTT CCG TAG CTG TTC AAG

     1                                2

1) restriction site, 2)  gene seqence

So two questions:

Primer design correct to insert a tag??

Will PCR work? b/c of primer length,annealing temp issues??

Would you suggest--breaking down the PCR--first only adding the tag and then adding the restriction site,kozak?

Thanks for any help!

You'll need some 4-6 bases of arbitrary junk DNA on the 5' of each of those primers to allow the RE cutting after your PCR reaction. The portion of the forward primer binding to the gene appears to be shorter than prudent. It'sl only 19 bases, compared to the 30 bp shown on the reverse primer.

Thanks for your reply. I was not too concerned about adding extra bases at the 5' end since I was going to clone it into a TA cloning vector for sequencing and then cut it out from there to insert into my expression vector.
I was concerned about the annealing temp differences for the primer set. Just to clarify- annealing temp should be similar for only the regions of the primer sequences that are complementary to the sequence- right?

jackster101 on Mon Sep 22 08:39:57 2014 said:

Thanks for your reply. I was not too concerned about adding extra bases at the 5' end since I was going to clone it into a TA cloning vector for sequencing and then cut it out from there to insert into my expression vector.
I was concerned about the annealing temp differences for the primer set. Just to clarify- annealing temp should be similar for only the regions of the primer sequences that are complementary to the sequence- right?

Yes, for the annealing step the important sequence for calculating the Tm is the part that corresponds to the gene's sequence.  After a few cycles with this tm you can increase it to the tm of the whole primer with the lowest tm if you want to but at lease for the initial cycles, stick to just the tm of the gene sequence.