HI!
I did not look into your primers but I just wanted to ask If you have tried with 64C as annealing temp.
I know that they ask you to design primers with Tm >/= 70 but you can still try first 7 cycles with 62 and rest 32 with 65 or so. I had the same problems but even 60-65C gave me specific bands.
Another way out is, you can add extra GC to 5' of your primers. I think adding 3-4 G/C will help. Just keep it in mind while reading your sequencing.
Both things work well for me!
HOpe it helps.
All the best!
Shifali
On Mon, 26 Jan 2009 Yarri wrote :
>Hej!
>>I am using a SMART RACE cDNA Amplification Kit from Clontech in order
>to get cDNA from roots of Datisca glomerata. I have been able to get
>few clones from cDNA I amplified with primers that had high Tm values
>- as recommended by the protocol. Unfortunately, now I have to work
>with more troublesome sequences, where best primers that can be
>designed have a Tm value <64C. I was trying to follow the protocol
>without much success. Does anyone has some experience with this kit or
>some suggestions how to approach the problem? I am new to RACE so I am
>not exactly sure how to troubleshoot RACE conditions.
>As an example:
>A sequencing result:
>CATGGAGGATGAGAAACAATATTACTAAAATAACTTTATGTTATGTGCTGTTTGAAGTTTATTTCTCTATGT
>TATAACTGCATGCTGCTATCGATCATTGTGAAACAAATCCTTATCAAATATTAATTATATATCTAGCCTTCT
>GACTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAMAAAAACAAA
>AAAACAAAACAAACAA
>>GSP2 primer I have designed: CACAATGATCGATAGCAGCATGCAG
>NSP2 primer: GTAATATTGTTTCTCATCCTCCATG
>Now I am waiting for additional primer that can be used as a nested
>one: GTAATATTGTTTCTCATCCTCCATG
>Thanks!
>> Marian
>P³aszczyca
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Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449
