promoter pcr using primers with restriction sites giving trouble - (Jun/09/2008 )

Hi all,

I have to clone my 2.5 kb promoter in PGL3 vector , the problem is the PCR amplefication . earlier I used the primers without restriction site to amplyfy my fragment and I got it amplified . But when I resdesigned them by putting Restriction site suddenly the primer is not working.

here's my primer sequence with and without restriction sites.

fwd(r site included) 5'-AGA-GCT- CGA- GGA- TTA- GGG- TTG- TAA- AGA- AGG- TTG- TGT- TC-3' (xho1 site)
rev (rsite included) 5'-GTG- TGT- GTG- TTA- AGC- TTG- CTC- ACC- TCT- CAT- TGT- AGT- T-3' (hind iii)

fwd (R site not included) 5'-GA- TTA- GGG- TTG- TAA- AGA- AGG- TTG- TGT- TC-3'
rev (R site not included) 5'-G- CTC- ACC- TCT- CAT- TGT- AGT- T-3'

the last pair showed the band at 62 and 65 degree temp. but with the pair mentioned first I cannot get anything all blank . wht can be wrong with my PCR ???

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Hi there,

Soryr to hear you're having troubles...are your primers for mouse or human? When I put them into the UCSC website, the primers don't match anything (with or without enzyme sites)

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Thanks for reply, my primers are zebrafish primers upstream to ptgs 1

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Well that makes sense I still don't get a match when I put in your primers to the zebrafish genome.
I wonder whether your primers are just too long?

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Your primers are not too long. Are you using the same PCR program with your new primers?

If you are, the problem may be your annealing temperature. The addition of the restriction enzymes not only increased your overall primer Tm, it also does not base pair with your template, initially. Calculate the Tm for the part of the primer that anneals to the template during the first cycle of PCR then calculate the total primer Tm for the rest of the cycles. So, you should use 2 different annealing temperatures, a lower temp (the original primer Tms) for 2 or 3 cycles, then the higher Tm (the new primer Tms) for >20 cycles.

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Either you got lucky in the first PCR or something has changed. There's no real reason why extra 5` bases should stop the PCR from working. Use the exact same conditions as the original PCR and check that you're not making any simple errors. If the sequence is GC-rich use some betaine.

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Thanks for the respons guys, yeah I know the Tm has increased thts why I was trying the temp range from 62- 74 but no luck.
I'll try to run touch down PCR as suggested hope that works for me.
I didnt use any DMSO or betain before . Do I still need to add it ? can I add DMSO?

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DMSO seemed to help my PCR problems when I added about it to be about 7% of the total PCR volume.

Also, another hint that was suggest to me and if you have the money in the lab is to amplify the promoter w/out the RE sites and topo clone it into an intermediate vector, or if money is an issue, you can TA clone it in and it is MUCH easier to amplify off of a plasmid than genomic DNA. I know an extra couple days of work, but it might help out a lot.

Then they even went one step further because some sites are hard to nip off the ends of a PCR amplified product and they TA cloned in promoter+RE sites and then used the RE to cut out of the TA vector and insert it into the final desired vector.

Just couple of hints I have learned along the way in the last couple of months.

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