i am trying to construct pGBKT-7 vector having my gene but i fail everytime. m using NdeI and NotI restriction enzymes and my gene length varies from 1600bp to 2000bp. i cut genes and vector using 37C in water bath for more than 8 hours and then ligate using 16C using vec:gene ratio (1:5, 1:4, 1:3) for 8-10 hours. when i run gel after colony PCR i get many bands and everytime company reply by saying that its empty vector. sometimes i get bands even only with water as negative control and i used new solutions but facing same problem.. i did restriction and ligation 4-5 times but failed.. any suggestions with this vector construction or others????
It is common to get false positives in colony PCR. This can be reduced by using vector specific primers to amplify the region. And if using gene specific primers, try to transfer the colonies to a new plate and then perform colony PCR with isolated colony, this way the template lying on the test plate is reduced.
@mangal Singh thanks for suggestion.. i am using isolated colony for PCR but still facing same problem. what do you think about restriction sites? is there any chance of inefficient cutting of these enzymes? should i change R.E and then try again r there is any other way to try with same R.Es??
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