I am trying BP reaction for gateway cloning for a long time, and getting a lot of colonies. but non of the colonies are positive.
steps
After adding complete attB1 and attB2 sites to insert, checking with PCR, I got bands on appropriate position. I am also using appropriate amount of Donor and entry vector. I have tried with different competent cells. I have also tried it with fresh lot of enzymes. Can anyone suggest how can I overcome this problem? I am struggling for more than 2 months.
The details:
Gene size: 966 Base Pairs
Donor: pDONR 207(gentamicin selection)
amount: 60 ng for both amplified product (insert)
250 ng donor. ....i am using equimolar concentration
water +buffer = to make up the volume
total reaction volume: 20 ul
BP clonase: 1 ul, 25 degree celsius incubation for more than 24 hours. r
reaction was stopped by adding proteinase k and incubation for 10 min at 37oC.
Cells used for transformation: DH5a.