Ligation Problem? Or Something else...? - (Jul/30/2010 )

I, along with another member of my lab, seem to be having some problem(s) with our cloning. I'm trying to insert a deletion mutant into a different vector and, based upon my digestion prior to ligation, the insert and the new vector are both absolutely fine. We use the protocol and kit from Qiagen for the DNA extraction/purification, so I don't see that being a problem, which leads me to believe that perhaps it is the ligation. One thing to note: for the initial digestion to get my insert and vector (using Not1 and Cla1), I've been using CIP (since that's what we have), but have not been heat inactivating it afterwards as per suggestion of another lab member whose had success in the past. Instead, I just heat inactivate at 65ºC for 20 minutes (to inactivate the REs; for the vector sample, this step is prior to the addition of CIP).

I've been trying to use T4 ligase (with the T4 buffer) with various ratios of insert to vector, 2:1.5; 4:1.5; 8:1.5; and 0:1.5 as a control. We make up 20 µL reactions so, for instance:
Insert 4 µL
Vector 1.5
ddH2O 11.5
10X buffer 2
Ligase 1

I keep the ligation reaction in a 16ºC water bath overnight. After the transformation, I see very few colonies on my control plate and I see a fair amount on the others (over 100), so that seems to be fine. However, after picking colonies, growing them up in LB+Amp, doing the miniprep, digesting the samples, and running my gel, I still only see my vector and I have yet to see the insert...

If you're cutting with NotI and ClaI, why use CIP at all?

HomeBrew on Sat Jul 31 01:06:52 2010 said:

Following this train of thought. Just drop the CIP step, CIP can be more trouble than it is worth. The ends of the vector are noncompetible. The plasmid will not religate if it has been completely double digested. If dephos is still desired consider using antarctic phosphotase. It if far more forgiving and it can be heat inactivated.

When using CIP, overdephosphoyrlation will render the molecule unligatable.

Also note, CIP can not be heat inactivated. If one where to follow protocol, 3' overhangs dephosphorylated by are incubated at 50 C with CIP. You will have to gel purify, column purify or phenol chloroform the sample to remove CIP.

perneseblue on Sun Aug 1 02:21:04 2010 said:

Cla1 can be a funky enzyme to cut with depending on methylation status...either you're not cutting the Cla1 site in the vector very well OR your ligated product is not being cut with Cla1...that would be my first guess...any other sites that you can use to verify insertion of your target?