what happens if I use T4 PNK buffer(NEB) instead of T4 ligation buffer (NEB) when phosphorylating

[ZQ]

 Hi everyone,

I didn't get any colony after cloning sgRNA into PX459(pSp cas9 BB-2A Puro). I uesd T4 PNK buffer(NEB) instead of T4 ligation buffer (NEB) when phosphorylating and anealing. Is this the reason?

Thanks in advance!

ZQ

[p.roma...@comcast.net]

Did you add ATP to the reaction? 

[ZQ]

No, I didn't add ATP.

But yesterday I repeated the whole cloning by using NEB T4 ligation buffer and again I got empty plates.

I did everything just as Zhang Lab's protocol said and I cannot figure out what could be wrong. Especially I suceeded before with PX461.

I am checking the vector by digestion with BbsI  just to exclude that Addgene sent me the wrong vector.

Thanks!

ZQ

[Ahmar Aziz]

I had the same problem when using the T4 PNK buffer during annealing because it does not have any ATP. I repeated the experiment with T4 ligation buffer and had successful clones. 

On Tuesday, August 26, 2014 4:58:50 AM UTC-4, ZQ wrote:

[Neuropath]

I use PX458 which is the same as PX459 except that it contains GFP instead of Puro. I didn't phosphorylate the annealed guide sequence but still got many colonies, 100% positives. You don't need to phosphorylate as the phosphate group on the 5' overhang of the BbsI cut vector will donate to form the phosphodiester bond during ligation. However, there will be a nick on both strands between the recessed end of vector and 5' overhang of annealed oligos. These nicks will be repaired by the bacteria when you perform transformation.

So it means the problem you are facing lies in other steps. ATP is very heat labile. If you have treated your ligase buffer badly like leaving it at room temperature for too long, it will stop working. Check your ligation and transformation with control vector and inserts.

[Anshuman Das]

Hey Neuropath
I am using Px334 vector and getting equal no. of colonies in control and ligation plate. I have tried upto overnight digestions. What could be the problem?

Thanks!
Anshuman

[SF]

  We have also been successful in ligation without T4PNK phosphorylation.  All you need to do is to order two oligo primers and do hybridization by heating a few minutes and slow-cooling down to RT, then dilute them in 100 times or so for ligation.  We constantly get a lot of clones and pick two or three clones and usually all of them are successfully sub-cloned.

 If you have no clone at all with this way, first you should be make sure of your primer design.  The primer hybridization usually ends up with in much excess DNA for ligation, so I think it is unlikely to be a problem.  So, especially if you have no background clone in control (without hybridization), I would suggest to check with your vector digestion and purification protocols  I think it still gives a fewer colonies in background even if the BbsI digestion ends are not compatible.

 If you do not use T4PNK, you should not de-phosphorylate vector since the nick needs to be fixed with that phosphate groups in the bacteria.

SF

[Alyssa RH]

Hi SF,

I am about to start the cloning protocol from the Nature paper. I see that you are suggesting that you do not need to use the T4 PNK enzyme when creating the oligos. My question is, if you do use the T4 PNK, are you using a dephosphotase on the backbone? The protocol suggests using phosphorylating the oligos and then there is no step to de-phosphorylate the backbone...I was planning to try with and without using the T4 PNK enzyme to see the differences. 

Also, I am wondering if anyone tried a different buffer than TANGO for the digestion/ligation step? I purchased the BbsI enzyme for NEB and the buffer is a bit different, but similar. Any thoughts or experiences.  

Best,

Alyssa

[Hanhan]

I wouldn't worry about NEB vs. TANGO. They both work well. 

For your ligation, you can do a kind of "proper" protocol, by using T4 PNK your annealed Oligos, and CIP+purified backbone. This way you can store the CIPed backbones for later use. 

Or you can do a "dirty-quick" protocol by adding freshly annealed Oligos to a freshly cut backbone reaction. Some people may not feel comfortable with this method, but it works just as well. 

Both methods are quite simple and easy for scaling-up. In my hand the key is to Anneal oligos on the day you do the ligation, and to keep a proper ratio between inserts: vector (follow the protocol) for your ligation.   

Good luck!

[Neuropath]

Hi Anshuman,

Sounds like you may be having a problem with your BbsI. Did you run a gel of the digest to make sure the vector is cut? If the digest had been complete, the vector should not be able to religate. The two ends are not compatible with each other. And you should gel purify the cut vector to remove the small released BbsI fragment.

~neuropath~

[ZQ]

Hi SF,

you were right and sorry for my late reply. I acutually did mistakes with my primers design. I got all my contructs done already weeks ago and thanks again for your advice!!

Best,
ZQ

On Monday, September 1, 2014 1:05:03 AM UTC+2, SF wrote:

[ZQ]

Thanks for your reply! I acctually did mistakes in primers design.
Learned something from you and you helped a lot!
thanks again!

[ZQ]

Thanks Hanhan,

your advices also helped a lot! I appreciate your reply!

ZQ

[Ram Ajore]

Hi all,

The problem to all this solution is just get 5'-end of the guide sequence phosporylated while sysnthesiszing it. It makes life easy and excludes some troubling steps.

i did so and it worked.

ram