Verifying Restriction sites generated in PCR of insert for subcloning - No colonies in transformation!Ligation issues?Insert issue? (Aug/07/2007 )

Hi All Lab genuises,

I am trying to subclone cJun in to PC DNA 3.1 vector. I have generated the cJun insert by PCR and added EcoR I and Xho I sites on the insert (or so I think). I have digested PC DNA 3.1 and insert with EcoR I and Xho I overnight at 37C. I have seen nice clean bands of right size on gel, so I proceeded to DNA ligation after gel extraction and purification.

I did DNA ligation at RT for 2 hours at vector:insert ratio of 1:1, 1:2, 1:4 with vector onlyand insert only controls. When I transformed I did not see any colonies that looked bacterial. i saw some funny looking colonies and when I grew them up in LB-Amp nothing grew.

I did not have a PC DNA 3.1 vector uncut control to know my transformation worked. But I am worried my problem is in the insert.

HOW CAN I VERIFY THAT I HAVE CREATED THE RESTRICTION SITES ON THE INSERT, BECAUSE CUT AND UNCUT RUN ALMOST SIMILAR ON GEL?

Appreciate any sugesstions.

If these digestion sites are designed as part of your primer you can be assured that they are there. PCR works by extending the primer that you designed and put into the reaction. However, did you make sure that you have plenty of bases on the edge for the enzyme to grab ahold? While EcoR1 isn't that picky, XhoI is. Look at page 243 in the current NEB catalog and you will see that with a three base overhang, after 20hrs of digestion only 75% of the product is cut. I also assume that you are using the EcoR1 buffer in the double digest. So, go back and check the sequences of the primer YOU RECEIVED...I've gotten bad primers before. All it would take is one base to be wrong. But if the digestion sequence is present in the primer, I assure you that the digestion site is present in the insert.

Now for the odd stuff, I have actually had ligations inhibited by gel purification of the insert.. don't ask me why. I once could not ligate an insert and it was suggested to me to go for the ligation without purifying the insert after digestion.. it worked. Also, ligations are inhibited by too much DNA. This is one of those times where less is more.

You could sequence the PCR products if you think there could be some errors. But usually it should be fine. Most importantly, you should verify if there are enough bases on the ends of the primers for adequate digestion and make sure you donot overdigest the DNA.

Good Luck !!!

You can ligate the insert alone, heat kill the ligase, and run the results on a gel. Ideally you will see high MW fragments. You can further clarify the situation by digesting the ligated product with each of the cloning enzymes one at a time, and both. The single digests should show double length fragments, while the double digest should show single length fragments. In reality, the single digests show a mix of double and single length fragments, and the ratio of these tells you the efficiency of ligation of the opposite cloning end. Use normal ligase, not the quick ligase, and heat kill the ligase. You can digest while you ligate if you adjust the NaCl concentration of the ligation buffer to 25 mM. and provide a brief 10 minute 37 post ligation digestion period prior to heat killing at 70C for 20 minutes.

You can also do a quick test of the ligase by ligating some DNA ladder quickly (most of them are just digests, anyway), and running a gel.