So frustrated... a question about blunt end cloning - (Dec/10/2005 )
I first tried to put a 1.2KB CDNA into 10KB retroviral vector, using blunt end ligation.
~20 colonies have right inserts, but they ALL are in wrong direction.
Then I tried to ligate an oligo into this vector with blunt end ligation, but they are all in one direction (13 clones).
Later, I tried to ligate that 1.2KB fragment into pBluescript (blunt end ligation), STILL, all 7 clones with insert are in SAME direction that I don't like.
I have tried several restriction enzymes, and used DH5alpha electro competent cells.
Has anyone seen this before? why my blunt end ligation seems to be directional?
Kinda frustrated and urgently need your help! Many thanks!
~20 colonies have right inserts, but they ALL are in wrong direction.
Then I tried to ligate an oligo into this vector with blunt end ligation, but they are all in one direction (13 clones).
Later, I tried to ligate that 1.2KB fragment into pBluescript (blunt end ligation), STILL, all 7 clones with insert are in SAME direction that I don't like.
I have tried several restriction enzymes, and used DH5alpha electro competent cells.
Has anyone seen this before? why my blunt end ligation seems to be directional?
Kinda frustrated and urgently need your help! Many thanks!
Dear freind,
Are you restricted to use blunt end vector only for your work? Because wrong oreintation in blunt end cloning is common. That why people using TA cloning vector.
Go and get one, I am sure that will be much more convenient.
Cheer
Best regards
Absolutely agree that blund end cloning makes us crazy in screening the direction. Other than TA cloning, there is a kit called Directional cloning.
For getting more colonies and cost concern , I like TA cloning kit.
Could it be that your gene in the "right" orientation is lethal?
It's also possible that you're really seeing just one clone, and that all the colonies are siblings of one another...
if blunt end cloning is not necessary better to do TA cloning
i think this will help
Thanks, guys. Someone told me this gene is not lethal, because they expressed a GST fusion in the bacteria. Unfortunately, the viral vector I am using has only 2 restriction sites, I guess I have to use PCR to introduce those restriction sites; or as you suggested, use TA cloning to ligate into other vector first. What do you think? Thanks again!
You can make your own "TA" cloning system very simply by incubating your insert with dATP only and Taq DNA polymerase and your blunt cut vector with dTTP only and Taq. After this just ligate.