Transformation /Cloning Problem - (Feb/02/2012 )

Hi guys!

I have been working for already a few weeks on my transformation/cloning reaction. However up till today with no results :-(

I have been trying to ligate a 1,5 kbp insert into a 8 kbp vector.

First of all I digest my vector and insert with Not1, afterwards I gel purify both.
Then I dephosphorylate my vector with CIP and gel purify this again.
Followed by a quick ligation (NEB) reaction with a 1:3 vector/insert ratio (5min at RT).
Afterwards I transform my SURE2 cells with 1 ul of the ligation reaction (in 10ul competent cells).

My positive control gave me good results however no colonies were seen on my experimental plates....

Can somebody please help me with this?

Thanks,

Alex

PS I have the intention that I lose quite a lot of product after gel purification... However I still have enough to start up a 3:1 ligation reaction...

Many possible problems. Tell us about the source of your insert. Is it a PCR product? Does it have 5' overhangs upstream of the cut sites? I would make sure that I purified a pcr before enzyme digestion. I would immediately stop gel purification of your vector prior to CIP treatment -- what is the point? I would switch from CIP to shrimp alkaline phophatase or antarctic phosphatase. I would make sure I heat killed the enzymes prior to ligation. I would switch from quick ligase to normal ligase buffer. Your gel isolation may lead to problems in DNA damage from UV. Use a long wave UV or better, a blue light for gel imaging.

Hi phage434,

The insert is not from a PCR product but from a cloning vector (pGemT easy). So you would suggest to not gel purify after the digest but directly go on with the CIP treatment? And at this moment, we only have CIP.... I already tried digesting 4 ug of my vector and reduce the UV exposure (max 5 sec) prior to gel purification... Any other things you can think of that might cause this problem?

Thanks!

Alex

Are you sure the SURE cells are highly competent. Ofcourse the positive control worked but is the vector (used for positive ligation) same or different compared to your actual ligation. Ensure CIP treatment is not too long as it can damage the vector.