Large MSCV Retroviral Vector Cloning --- Help Needed! - (Sep/17/2008 )

Hi everyone,

I'm currently trying to ligate an insert into a MSCV retroviral vector, followed by transformation. However, this hasn't been working for about 2 weeks of attempt.
This is my scenario:

After an overnight ligation at 16 degC (Using a 5:1 insert - vector ratio, from Promega's biomath calculator), I transform the ligated DNA into Stbl3 E.coli competent bacteria from Invitrogen, following the protocol given.

However, I get very few colonies (around 3-4 colonies a plate), and after doing a miniprep and digesting with 2 unique restriction enzymes to check for the correct size of the insert, i get different band sizes on the gel for each of the different colonies.

As my final ligated product is about 10kb large, I think this could contribute to the small no. of colonies.
As for the different bands, I'm suspecting high recombination due to the LTRs in the retrovirus.

I would like to ask if anyone has been through this scenario of cloning large retroviral vectors, and is there anything I can do to increase the efficiency? eg. use a diff strain of bacteria, diff conditions etc.

Thank you for your help! =)

I clone viral vectors this large (>10kb) with good success and lots of colonies. I use BL-21 cells, and ALWAYS do a control (vector alone reaction) in parallel. I also use a quick ligation kit from Roche instead of ligating overnight, and directly transform the ligation mixture into my cells.

How does the number of colonies on your control plate compare to your experimental? Also, how much are you plating afterwards?

Ginger

Do you have clean bands for your insert PCR product? How are you cleaning up that reaction? Is this a blunt or cohesive ligation? Do you run a no-insert control transformation?

Could you give a more detailed description of the cloning steps and enzymes involved.