2007 )

Hello everybody,

I want to amplify the whole CDS which is 1281bp. .my template is cDNA,please see the attachment. The cds is 1592-2872. So i want to amplify a product of 1501 bases which includes the CDS.GC% of product is 61% . forward primer: AGGACCAAACGATCTGCG, Tm56.2. reverse primer:GAACAAGACAAGCGGGACA, Tm 56.3. 94 3min (94 1min annealing 50s 72 1min40s) 30cycles 72 10min . annealing temperature 52.7,54.6,57.4,59.8. I can not observerd any product band except house keeping gene actin.

The attachment is the product sequence of 1501 bases,and CDS is from 1592 to 2872. I have tried many times ,but they all failed. I can not find what is wrong, can anyone help me?Thank you very much.
gctttttcct ccctttagta gctcagccta acgtctttcg tttttttttt
1141 tttttttttg cccccgagga tcttccatgt aggaagccga ggctggcgag cccgacactc
1201 gggagccact gtaggggggc cttttttggg ggaggcgtct accggggttg cctcggccgc
1261 ccccagggaa gcggcggccg cgttcctcca gggcacgccg gggcccgaaa gccgcgcagg
1321 gcgcgggccg cgccgggtgg ggcagccgaa gcgcagcccc ccgatccccg gcaggcgccc
1381 ctgggccccc gcgcgcgccc cggcctctgg gagactggcg catgccacgg agcgcccctc
1441 gggccgccgc cgcttctgcc cgggcccctg ctgttgctgc tgtcgcctgc gcctgctgcc
1501 ccaactcggc gcccgacttc ttcatggtgt gcggaggtca tgttcgctcc ttagccggca
1561 aacgactttt ctcctcgcct cctcgccccg catgttcagg accaaacgat ctgcgctcgt
1621 ccggcgtctc tggaggagcc gtgcgcccgg cggcgaggac gaggaggagg gcgtgggggg
1681 tggcggcgga ggaggcgagc tgcggggaga aggggcgacg gacggccggg cttatggggc
1741 tggtggcggc ggtgcgggca gggctggctg ctgcctgggc aaggcagtcc gaggtgccaa
1801 aggtcaccac catccccatc ccccaacctc gggtgccggg gcggccgggg gcgccgaggc
1861 ggatctgaag gcgctcacgc actcggtgct caagaaactc aaggagcggc agctggagct
1921 gctgcttcag gccgtggagt cccgcggcgg tacgcgcacc gcgtgcctcc tgctgcccgg
1981 ccgcctggac tgcaggctgg gcccgggggc gcccgccagc gcgcagcccg cgcagccgcc
2041 ctcgtcctac tcgctccccc tcctgctgtg caaagtgttc aggtggccgg atctcaggca
2101 ttcctcggaa gtcaagaggc tgtgttgctg tgaatcttac gggaagatca accccgagct
2161 ggtgtgctgc aacccccatc accttagtcg actctgtgaa ctagagtctc cccctcctcc
2221 ttactccaga tacccaatgg attttctcaa accaactgca ggctgtccag atgctgtacc
2281 ttcctccgcg gaaaccgggg gaacgaatta tctggcccct ggggggcttt cagattccca
2341 acttcttctg gagcctgggg atcggtcaca ctggtgcgtg gtggcatact gggaggagaa
2401 gactcgcgtg gggaggctct actgtgtcca agagccctcc ctggatatct tctatgatct
2461 acctcagggg aatggctttt gcctcggaca gctcaattcg gacaacaaga gtcagctggt
2521 acagaaagtg cggagcaaga tcggctgtgg catccagctg acgcgggaag tggatggcgt
2581 gtgggtttac aaccgcagca gttaccccat cttcatcaag tccgccacac tggacaaccc
2641 ggactccagg acgctgttgg tgcacaaagt gttccctggt ttctccatca aggcttttga
2701 ctatgagaaa gcctacagcc tgcagcggcc caatgaccac gagttcatgc agcaaccatg
2761 gacgggtttc accgtgcaga tcagctttgt gaagggctgg ggccagtgct acacccgcca
2821 gttcatcagc agctgcccgt gctggctgga ggtcatcttc aacagccggt agtcggtcgt
2881 gtggtgggga gaagaggaca gggcggatcg tgagccgagc aggccaccgt tcaaactact
2941 tgctgctaac ctttcccgag tgattgcttt tcatgcaaac tctttggttg gtgttgttat
3001 tgccattcat tgttggtttt gttttgttct gttctggttt gttttttttt ttttttcctc
3061 ctcctttctc gtcatccgtg tgtcccgctt gtcttgttct ttgagaaatt agcttatggt
3121 gcggattttt gttgggttgt gtgtgtgtgt tttgtttttg ttttgaggtg gtgggtgtgg
3181 ttggcaggac accccctccc cccatatacg aagacaggaa acgagagtca gcactgccaa
3241 gcatggtgtg tgaaagtggg caccaccttc cctttggatc agcgtttcgg ttgtccgtgc
3301 gtaggggtgt acccgagcga cagatggggg aagtgctttt ttgtgtgtgt gttctttatg
3361 gatgcccccg gc

-wing111-

Hi Wing111,

I have attached a file with the sequence that you provided with the CDS boundaries marked + primer sequences.

It appears your reverse primer isn't in the CDS sequence, so if your using cDNA as your template you wouldn't get a product.

-JessH-

QUOTE (JessH @ Nov 4 2007, 06:41 PM)

Hello JessH,
First,thanks for your reply.I want to amplify the whole CDS and have tried to amplify several products including CDS.I designed several primer pairs by primer 5.0. The chosed one is suitable to my purpose.Theoretically,a product of 1500bp could be amplified,but nothing was observed under UV except house keeping gene actin beta.
I divided the CDS into two parts,one is 1529-2240,the other is 2214-2908.Although reverse primer of the second one(2214-2908) isn't in the CDS sequence, it could be amplified and then cloned. But the first one(1529-2240) couldn't be detected.Maybe GC% is too high about 68.3%.When amplify the 1500bp including CDS,GC% is about 60.3%,so someone advised me to amplify it.But ,I get nothing.

I don't understand why primer isn't in the CDS sequencd,I couldn't get a product.THE sequence is also is exon area.Could you tell me?
Any advice is deserved to be reverent.

-wing111-

With such a high GC% you probably need to include an additive in your PCR to help dissociate the template. Look up betaine - it is excellent. Make up a 5M solution and use at a final concentration of 0.5 - 2 M.

Good luck, Rob

-killerkoz17-

It seems that your primers are fine then.

I agree with Rob, definately try betaine! Sigma sell it pretty cheap, you need to make sure you buy PCR grade:
http://www.sigmaaldrich.com/catalog/search...ail/SIGMA/B0300
A final concentration of around 1 M should be fine - but you can optimise for your reaction.

Also I use KlenTaq polymerase for GC rich/secondary structure prone templates. Its a blend of Taq polymerase plus a proofreading enzyme so it has higher processivity. Its also from Sigma - I swear i dont work for them!! But try the betaine first.

Cheers, Jess

-JessH-

Thanks all of you! I'll have a try.

-wing111-