Hello,
I'm trying to identify the unknown gene (phosphoglycerate mutase) from a nematode by RACE technique (Clonetech, Takara)(The CDS orthrologue gene in C.elegans is 1.5 Kb ). I performed 5' and 3' RACE reaction together with 25 cycles (annealing temp. 65C; kit recommended 68C)and then observed on 1.2% agarose electrophoresis. 3' RACE product gave a 600 bp size but 5' is not presence. Then, I performed the 5' RACE reaction with 5 cycles (total 3c cyles). The result showed approximate 10kb of 5' RACE product size (In figure). So, i want to know "Is possible that the nematode gene has 5'utr up to 10kb?" or it's the error on electrophoresis.
Thank you so much,
Sarit
Is that the first round PCR product? Anyway It doesn't look like a specific band to me. BTW you will never get a single band from RACE PCR. Although the kit recommend annealing Tm is 68c, but it also depends on your GSP. Use this cal. 4(G+C) +2(A+T) - 5 to work the Tm of your GSP. I would suggest you to try the touchdown PCR. For example, if your GSP Tm is 63c, Try
Step1
94c 4min
step 2
94c 30s
67c 30s (-1c/ cycle for 5 cycles)
68c 3-4 min
Step 3
94c 30s
63c 30s (for 30 cycles) or even 35 cycles
68c 3-4 min
Step 4
72c 15min
Good luck
1-Make triplicates of this reaction and see whether you get the same results.
2- Optimize your annealing conditions and observe whether you get the same size product.
3- If you consistently obtain the same results, Purify this product and PCR it with newly designed internal primers to see if you are amplifying your target sequence.
Good luck
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