cloning trouble - (Mar/17/2009 )
Will try that!! thnks for the comments..
Even though the question has been answered I feel I should mention TOPOXL vector as its never failed me, even when I can barely see the PCR product. It contains a lethal gene that is inactivated by the insert so you get no vector background and Topoisomerase instead of ligase. I use Pfx which has good proof reading and no taq then add 4u Taq at 70oC (as said below) for 5 mins to the PCR tube to add overhangs. I gel extract using either EtBr or crystal violet (gel extracting also removes any template plasmid which may give colonies on the plate) and do the 5 min topo reaction.
LP2 on Mar 20 2009, 01:48 AM said:
Would you mind explaining the words in bold? do you mean that other than your desired PCR products, there were other stuffs that ligated to the vector?
the kit recommends a certain concentration of pcr products for ideal vector-product ligation.. i think lp2 meant that he got the req'd reacn even when the eluted pcr product was of low conc..
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
LP2 on Mar 22 2009, 11:37 PM said:
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.
