no pcr amplification product - (Sep/14/2009 )

Hi, i really need some help with a round of SDM that i am doing.I am not getting any pcr product at all.

i have changed pretty much everything that i can think of:
-changed polymerases
-changed all buffers, dNTP's etc
-used 2 different pcr cyclers
-used touchdown for the annealing temp and the extension time
-redesigned primers x 2

i have 3 sets of fwd and rev primers, all 27 bp long TM high, ranging from 71 to 76, but this is not unusual for our lab, we regularly use such primers, GC% 49- 55%,

pair 1 - CAC GGT GGC CAA ACG GAA ATA GGA CAG
- CTG TCC TAT TTC CGT TTG GCC ACC GTG


pair 2 - CAC GGT GGC CAA ATA GAA ATA GGA CAG
- CTG TCC TAT TTC TAT TTG GCC ACC GTG

pair 3 - CAC GGT GGC CAA CTT GAA ATA GGA CAG
- CTG TCC TAT TTC AAG TTG GCC ACC GTG

the template DNA is 7KB long and is regularly used, the control works (T7 and BGh rev)

i have shortened and lengthened all 3 sets of primers and still have no product. i have re-prepared the template DNA too

i run a small amount of pcr product with DPnI digested product on a 1% agarose gel and i see no bands in the digested and a very faint band in the undigested (template??). i have even gone onto to transform competent cells just incase and i get no colonies.

I have only been doing pcr for 18 months and i am sure i am missing something but just can't focus on what it is? please can somebody help?

Seems to me that the culprit could well be the Taq. 7kb can require a really good polymerase to span that length. When you said you "changed polymerase" did you mean you tried a new vial or a whole new enzyme?

glenk on Sep 15 2009, 06:35 PM said:

I tried both platinum taq hifi and pfu turbo, which I use on a regular basis for plasmids of this length without problem.

I have just had a last ditch attempt where I've increased pretty much every variable! Denaturation upto 45 s, annealing - 45 s and extension times - 15 min. I also prep'd another set with double the amount of primer, dNTP's, and polymerase. I have got weak bands with "normal" amounts of pcr reactants but stronger bands with the increased reactants. I have some colonies after transformation and will know if they have worked next week when the sequencing is returned.

hopefully it has worked, but I am still in the dark regarding the actual problem, but i'll look into that at later (maybe)!

Hi lsb5579,

You mention that your control works.

It seems that the problem now might could be your DNA extraction. Perhaps your template DNA had been fragmented or degraded and that explains 7kb length hard to be amplified. Try use a more suitable method and see whether it works.

Good Luck.


Adrian

Thanks for the suggestion, however I did use an existing plasmid and also reprep'd the same plasmid, when they did not work, i arranged for a sample from another lab, which didn't help either. fingers crossed for the colonies i've managed to get anyway!

Some things to think about:
* you should be using Pfu Turbo or Ultra or other non-displacing enzyme for this reaction. Taq and Taq mixtures will not work.
* You should check your template to see if there might be regions of very low GC content. For these templates, you need to lower the extension temperature and slightly extend the extension time. Try 65C extension and 9 minute extension.
* I would immediately try a short PCR reaction using your same reagents and some primers for a 1Kb reaction just to check that your template and PCR conditions were correct.
* How much template are you using?
* How many cycles are you doing?

phage434 on Sep 17 2009, 03:57 AM said:

Hi, thanks for all the input.

I have been using pfu Turbo, i used 50ng of template, which i eventually up'd to 100 ng. i have done 30 cycles.

i have a new problem, i now cannot get it to sequence very well with my BGH Rev. there is a PolyA tail at the 5' end of my construct, 150 bp after my stop tag. i am now designing some internal primers.

thanks again

lsb5579 on Oct 1 2009, 01:51 AM said:

Try 40 cycles, occasionally it may be solution for the low-rate reactions.

And check your DNA dilution. To more dilution may be as bad as no dilution at all (in the first case there are not enough DNA templates and in the second there to many reaction inhibitors such as polysaccharides )

hey isb5579, what a great name, looks like a code.

the problem i felt in ur primers is that sequence of one primer is complimentary to other primer in all the sets (when matched as per their 5'-3'sewuence), so how can it bind to ur template DNA when they will bind themself in PCR reaction. so seems difficult to amplify what u want

check ur sequence was:
pair 1 - CAC GGT GGC CAA ACG GAA ATA GGA CAG
- CTG TCC TAT TTC CGT TTG GCC ACC GTG


pair 2 - CAC GGT GGC CAA ATA GAA ATA GGA CAG
- CTG TCC TAT TTC TAT TTG GCC ACC GTG

pair 3 - CAC GGT GGC CAA CTT GAA ATA GGA CAG
- CTG TCC TAT TTC AAG TTG GCC ACC GTG

and yes all ppl: Plz, Do tell me if I guessed it Rite or Rong

Hi, 2 out of 3 of my pcr reactions have eventually worked, awaiting results on the 3rd.

Hi microbes, i think you are comparing the 5'-3' of one primer against 3'-5' of the other. They are reverse and complimentary sequences (as i have designed them to be) of each other.

there is a big question over the polyA tail and also i have just arranged to have my pcr machine temp validated, i think maybe it is out of sync (for various other reasons!!!)

thank you for all your suggestions, fingers crossed for the other 15 that i have to do now!!!!