help TA cloning!

manojmadhav at hotmail.com manojmadhav at hotmail.com
Sat Dec 14 14:39:26 EST 2002


Hi All!
i write this to seek your help for my  problems with pBADThioTOPO vector from invitrogen.

the vector is 4.4kb and the insert am trying to insert is 2.4 kb. i ligated straight from PCR reaction mix(Taq amplification), as well as with gel purified PCR band. in the latter case i get only few colonies, while first one gives plenty.i got a lot of transformation after ligation, but all appeared to be of the size of vector to vector ligation. but further analyses showed that its not vector to vector ligation. there is also an extra low intensity(<5%) high molecular size band in my PCR product. but i wonder how a low quantity large size band can surpass a 
high quantity(95%) small size band to liagte to vector! I have screened around 300 clones from foue ligation reactions done in different ways, but of no avail!

i also tried to ligate the company-supplied control, which worked well as evidenced by blue-white selection. I simply dont understand why my insert is not getting ligated, while the control template and primers supplied by manufacturer but amplified by myself using the same conditions as with my isert is doing well.
i wish someone would have something to share with me to save myself, and probably to others who have same problem..
best regards
Manoj N K
Yale university

http://biowww.net/mynews/tree.php?group_name=bionet_molbio_methds-reagnts&begin=0



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