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LP2, on Mar 22 2009, 11:37 PM, said:
In response to to the above post by jiajia1987. There were two instances of this.
1) when I loaded 40ul of PCR reaction onto a gel I could just see a faint but discreet band at the desired size. I then used the remaining mix to do the TOPO reaction and got lots of colonies. I screened them and found that they were all correct.
2) When looking into virus splicing patterns I used a sequence specific oligo and an oligoT to make cDNA of the region of interest. I amplified it for about 5 cycles and TOPO cloned it. This gave colonies, some of which contained junk but 4 or 5 had sequence which told me that my splice site and polyA site were working. (i'm sure there are better ways of doing it but I just used the materials that came to hand and was confident with the result).
Hope this is clearer
1) when I loaded 40ul of PCR reaction onto a gel I could just see a faint but discreet band at the desired size. I then used the remaining mix to do the TOPO reaction and got lots of colonies. I screened them and found that they were all correct.
2) When looking into virus splicing patterns I used a sequence specific oligo and an oligoT to make cDNA of the region of interest. I amplified it for about 5 cycles and TOPO cloned it. This gave colonies, some of which contained junk but 4 or 5 had sequence which told me that my splice site and polyA site were working. (i'm sure there are better ways of doing it but I just used the materials that came to hand and was confident with the result).
Hope this is clearer
Dear LP2, thanks for the reply.
