11 replies to this topic

[Thapa]

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

[ranvi]

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

[allynspear]

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.

[Thapa]

ranvi, on 07 October 2011 - 11:32 AM, said:

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

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

[Thapa]

allynspear, on 11 October 2011 - 08:33 AM, said:

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.

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

[allynspear]

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.

[phage434]

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

[Thapa]

allynspear, on 18 October 2011 - 07:27 AM, said:

if you are digesting your annealed oligos or if you have annealed oligos with overhangs?

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 18 October 2011 - 07:27 AM, said:

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.

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).

Edited by Thapa, 19 October 2011 - 02:13 PM.

[Thapa]

phage434, on 19 October 2011 - 05:00 AM, said:

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

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???

[phage434]

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.

[allynspear]

If you are just EtOH ppting your cut oligo, there is a good chance that the ends of the ds oligo are interfering with your ligation.  Each end will have a compatible overhang with your vector on one side, and a blunt end on the other.  These little ds oligo "nubs" can just act as a cap to your vector, preventing proper ligation.  However, since you are cutting your oligo, then all fragments (the overhangs of both ends and both sides of the insert) will have 5' Phosphates.

I do understand that you cut your vector with 2 different enyzmes, and so you think you don't need to phosphatase treat your cut vector.  This is a mistake that a lot of people make.  There is a difference between what theory tells you is supposed to happen versus what reality tells you actually does happen.  Unless you perform a vector only ligation and transformation and get ZERO colonies on a selection plate, then there is always the risk of vector self ligation.  Here is how this happens:

(1) A double cut vector, cut in the MCS, looks the same on a gel as a single cut vector.  Even 10 molecules of single cut vector that you can't see will religate and transform.
(2) Single stranded overhangs are always subject to shearing or degradation, so incompatible overhangs do not guarantee zero ligation
(3) Self-ligation is always SIGNIFICANTLY more efficient than bi-molecular ligation, so Vector Only will always be favored over Vector+Insert

Therefore, if you give it the opportunity, even just a few molecules here or there, Vectors will religate on themselves and give rise to colonies on your transformation plates.  This is why many people phosphatase treat their vectors, even if they should have incompatible overhangs.

If you were to start all over again, I would redesign your oligos so that you would generate the overhangs that you need by annealing, rather than digestion.  I would be sure to add 5' Phosphates to the oligos, and Phosphatase treat your vector.  If you don't want to re-order your oligos, you can try to gel purify your cut dsOligo on a TBE-PAGE gel or high-percentage agarose gel, as long as your extra cut ends are not similar in size.  The TBE-PAGE gel will give you the best resolution and yield, but if you aren't set up to do that, then a high percentrage agarose gel (2.5%-4% NuSieve should work) is your next best bet, but just be sure you digest a lot of the oligo because your gel purification yield from QiaQuick on a 50 bp band will be fairly low.

Best of Luck.

[Thapa]

allynspear, on 20 October 2011 - 04:08 AM, said:

If you are .................... QiaQuick on a 50 bp band will be fairly low.

Best of Luck.

Thank you so much allynspear. I appreciate your knowledge and detail answer...its really worthful for the beginers like me.

Thanks a ton
Thapa