Selection of clones after ligation? - (Oct/07/2011 )

Bioforumers,
I did a double digestion (NheI and BglII) of my luc vector as well as synthetic oligo. I then purified the vector by gel extraction-qia quick method where as the oligo insert ppted with sodium acetate/etoh (coz my insert is <50bp). Next was ligation: Vector+ligase (control) and vector+insert+ligase for 10 min at 25C.

After tranformation, I didnt see much changes in the number of colonies on LB agar (+amp)??

I got abt 40 to 50 colonies in each plate. Should I go for selection picking these colonies??

Since I am new at cloning, what screening method is appropriate for easy hints??

or I should start with scratch??

I will appreciate your time.

Thanks,
Thapa

yes screen about 5-10 colonies, mini prep them then do the digestion look for the correct size then go for sequencing if they are the right clone sequence will give u the answer

I agree that you should go ahead and check a few colonies, but having the same number of colonies on vector only and vector+insert is not a good sign. I would try changing your ligation conditions to 16 degrees overnight and see if that helps. Also, are you quantitating the amount and ratio of vector and insert? You should ideally have a 3:1 molar ratio of insert to vector. Lastly, if you keep having vector background, then it is possible that you didn't get 100% double digestion with you vector and you can try to phosphatase treat your vector, but only if your oligo is 5' phosphorylated. If it is not, then this may also be reducing your cloning efficiency.

Best of Luck.

ranvi on Fri Oct 7 19:32:38 2011 said:

Thanks ranvi, i am really worried with the vector background.

allynspear on Tue Oct 11 16:33:26 2011 said:

Hi allynspear,
Some of my concerns match with you...though im really a fresher in this area. When I used two RE, though i have got shifted bands on each and on double digested band (when i ran on gel)...it is given on neb that the RE on buffer 2 have 75% and 100% efficiencies. This means, there were probably single digested vector and those religated (as i didnt use phosphatases) and gave me higher background?? what you say??

any troubleshootings?? I wanna start something fresh again rather than random picking forever....hehe

It's hard to visualize what you're seeing without actually seeing the gel. As far as troubleshooting/starting fresh, I did want to know if you are digesting your annealed oligos or if you have annealed oligos with overhangs? If you are digesting them, I am wondering how much extra sequence do you have before/after your RE sites in the oligo?

As far as the vector goes, I would definitely phosphatase treat the final gel purified vector with a heat inactivate-able phosphatase like Shrimp Alkaline Phosphatase or Antarctic Phosphatase. At least this will limit the amount of screening you are doing.

Best of Luck.

Just checking -- the annealed oligos have a 5' phosphate?

allynspear on Tue Oct 18 15:27:19 2011 said:

I have ordered ssOligos and then annealed equimolar ssOligos to obtain dsOligo. Then, they were double digested with REs. As these oligos were <50 bp, I didnt gel purified instead followed Na-acetate/EtOH pptation.

allynspear on Tue Oct 18 15:27:19 2011 said:

Because I digested my vector with 2 REs simultaneously, I didnt use any phosphatases. Gel purification was done with qiaQuick and after confirmation by running gels (1. uncut vec, 2. RE-cut vector, 3. RE-cut vector, and 4. double REs-cut vector).

phage434 on Wed Oct 19 13:00:20 2011 said:

I have small oligos (insert for luc-vector) which I have digested with the same enzymes that I used for my vector (PGL3 basic). As I told you I am new in this area, I dont know if the cut insert has 5' phosphate or not???

Are there 5' overhangs on your annealed oligo fragments sufficient to allow the restriction enzymes to cut? Perhaps you could simply give us the sequences of the two annealed oligos.