help in PX330 plasmid ligation

[yuanwu liu]

Dear all menbers,

     We recently ligation a gRNA into PX330 but all failed following protocol given in forum. Our step as follow:

First Round ligation:

1.digest 5ug PX330 and gel purity with kit the finnal concentration is 20-30ng/ul

2.Phosphorylate and anneal oligos 100uM

3.50ng digested PX330 with 1ul undiluted annealed oligo duplex to ligation in T4 ligase following ligation protocol

4.transformation

sequencing with human U6 primer and all picked clone is PX330 backbone.(we thought it might be undigested plasmids contamination in gel purity)

Our BbsI was stored in -20°C for severeal months, after digenstion PX330 it appeared two bands.we cut and purified the upper one (left picture is lower band).

After that we picked 3-4 clones mixed in one tube to sequencing found it's ligation efficiency is very low, we tested 4 mixed sample just one is correct.

To improve ligation efficiency we performed next ligation:

1.Following new version protocol, perform digestion and ligation in one step (this method I used before and it's works well)

2.This time we changed a new BbsI and T4 Ligase from NEB. Reaction buffer was prepared by NEB recommend:buffer 2.1 with 1mM ATP

3.before ligation we check if 1ul BbsI can digest 100ng PX330 find it's okay to totally digestion within 1h.(left with three lane:1kb ladder/PX330/digested px330)

4.After ligation we picked 10 clones for sequencing but all backbone plasmids too.

I was confused why our ligation efficiency is relatively low? who can give us a suggestion?

I found there are a subtle difference between two version protocol, in old version protocol oligo duplex concentration was 100mM but new version was 100uM does this important to ligation results? I caculated it's mol ratio to plasmids we used in ligation is around 1:40. We use 100mM oligo duplex to do ligation before and it's efficiency is very high nearly 90%.

[A.Moosdijk]

My ligations thus far worked very efficiently for pX330. 

In your first try, you use undiluted annealed oligo. I dilute my 100 uM oligos (after annealing) 1:400 in 0.5x annealing buffer and use this to ligate with ~20 ng of open pX330. Maybe your ratio oligo - pX330 is too high? 

This could then also be the case in your other protocol.

And did you add the overhangs (CACC and AAAC) to your oligos? 

Anoeska

[yuanwu liu]

Thanks for your help, we try to ligation oligo without dilution but also useless. this morning I doubt my oligo overhangs but after check again it's OK(CACC/AAAC was added). we checked our T4 ligase activity by gel electrophoresis ligation product results as follows:

lane1,PX330+BbsI

lane2&3,PX330+BbsI+ligase; 

lane4,PX330;

lane5,1kb ladder.

在 2014年4月30日星期三UTC+8下午4时24分59秒，A.Moosdijk写道：

[Elitsa]

hei,
I think Anoeska gave you very good suggestion - recheck  the concentrations of your oligos and the ratio of the oligo/vector for restriction/ligation. Also, check the proper annealing of your oligo. You have sequenced your vector, right?

We have already used several times very efficiently the protocol from F.Zhang group (http://www.ncbi.nlm.nih.gov/pubmed/24157548) with  very minor modifications - we did not phosphorylate the oligo. Briefly, we annealed of the oligos (100 uM each in Tris-HCL buffer/H2O), checked the annealing on the 10% native polyacrylamide gel and performed the simultaneously digestion and ligation using 1:200 diluted (in H2O) annealed oligo and 100 ng Cas9 vector for 2 h at 37C...Then usual transformation in bacteria and plenty of colonies on the next day...all picked, all positive.

hope this helps a little,  good luck:-)
Elitsa

[yuanwu liu]

hi,

 

   Elitsa,Thanks for your reply, after recheck my oligos concentration I found it was my mistake. I will try diluted oligos again.Thnaks again for your and Anoeska suggestions.

Yuanwu Liu

在 2014年5月1日星期四UTC+8下午8时13分30秒，Elitsa写道：

[Fatwa Adikusuma]

For your first version I would say make sure you digest the Px330 completely with Bbs1. I used to religate linearized PX330 alone as a control and transformed to competent cells and It hardly gave me colonies which means incompatible overhangs of linearized PX330 can not easily religate.

Cheers,

Fatwa

[yuanwu liu]

Fatwa,

Thanks for your suggestion, indeed I always doubt my digestion not completely in my first experiment. But the most notable concern was that why it cuts one of two restriction site in condition there are no differnecs between them? I've nenver meet this before. 

在 2014年5月5日星期一UTC+8上午11时51分23秒，Fatwa Adikusuma写道：

[yuanwu liu]

Hi,

    After dilution my oligos it works well and all clones are positive. I wanted to know why oligo/vector ratio is so important in ligation, if I added much more oligo in theory it maks more possibility to ligation into my vector?

thanks for all helps

Yuanwu Liu 

在 2014年5月1日星期四UTC+8下午8时13分30秒，Elitsa写道：

hei,