ligation problem - PCR, RT-PCR and Real-Time PCR

am trying to clone a gene of 1.2kb into pGEX4t-3 vector by PCR cloning with sal1 and BamH1 enzymes. after pcr, i gel purify the product, double digest purify and run on the gel, i see a faint band around 3 kb.
i went ahead with ligation with this product.

iset up ligation at 1:3; 1:5 ratio, i see the colonies only in the vector plate but in the V+I plate.

i had cloned this gene in to two different vectors with no problems.

can any one offer me any help

thanks
aparna

aparna on Feb 10 2010, 05:03 PM said:

am trying to clone a gene of 1.2kb into pGEX4t-3 vector by PCR cloning with sal1 and BamH1 enzymes. after pcr, i gel purify the product, double digest purify and run on the gel, i see a faint band around 3 kb.
i went ahead with ligation with this product.

iset up ligation at 1:3; 1:5 ratio, i see the colonies only in the vector plate but in the V+I plate.

i had cloned this gene in to two different vectors with no problems.

can any one offer me any help

thanks
aparna

hi aparna, one possibilty could be that your enzymes are not working fine. Did u try single digestion with each of these REs and checked their efficiency? your insert might not have got digested properly........did you put the digestion for the vector and insert together, and then checked if the vector is completely linearized??? and maybe, you need to have more concentration of your insert....

aparna on Feb 10 2010, 05:03 PM said:

am trying to clone a gene of 1.2kb into pGEX4t-3 vector by PCR cloning with sal1 and BamH1 enzymes. after pcr, i gel purify the product, double digest purify and run on the gel, i see a faint band around 3 kb.
i went ahead with ligation with this product.

iset up ligation at 1:3; 1:5 ratio, i see the colonies only in the vector plate but in the V+I plate.

i had cloned this gene in to two different vectors with no problems.

can any one offer me any help

thanks
aparna

hi aparna, one possibilty could be that your enzymes are not working fine. Did u try single digestion with each of these REs and checked their efficiency? your insert might not have got digested properly........did you put the digestion for the vector and insert together, and then checked if the vector is completely linearized??? and maybe, you need to have more concentration of your insert....

ligation problem - (Feb/10/2010 )