RACE troubleshooting

Rinaldo C. Bertossa r.c.bertossa at rug.nl
Fri Mar 4 10:52:11 EST 2005


Hello,
I am trying to get (RACE) the 5'- and 3'-transcripts-ends of my gene.
TROUBLE: I do not manage to get clear bands (only smear).

Background information:

1)A Northern blot was made before:
- 5ug polyA+ RNA (= the gene is minimally transcribed)
- radioactive labelled gene-specific 300bp-fragment X (the only
gene-information I have)
- Result: I get 4 clear bands ranging from 6 to 20 kb (differential
transcription?).

2)RACE Kit: FirstChoice RLM-RACE kit (Ambion):
- with polyA+ RNA: 250 ng for 5'RACE, 50 ng for 3'RACE.

3)PCR kit: Expand Long Range PCR System (Roche): (procedure as
described in kit)
- 1 ul cDNA (from above) used in nested PCR reactions;
- Cycling:
1x 93C, 2'
10x [93C, 20''- 60C, 30''- 68C, 2'30'']
30x [93C, 15''- 60C, 30''- 68C, 2'30''+10''/cycle].
- I also tried longer extension times (8').

4)Positive controls: I do amplify the fragment X form the cDNAs (after
40 cycles in a standard PCR reaction).

ANY suggestion is welcomed.

Thank you in advance,
Rinaldo



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