1. I assume you've checked your oligo design so that they have the correct overhangs? (CCGG.....TTTTTG for the forward and AATTCAAAAA... for the reverse?)

2. How are your oligos purified? This is important.

3. what buffer are you using to anneal your oligos?

4. Are you doing a simultaneous vector digest or a sequential vector digest?

5. If you are gel-purifying your digested vector with ethidium bromide staining, be sure to use long-wave UV and minimize UV exposure time

Hi, The answers are as follows:

1. The overhangs are correct.

2. QC is done by mass spec/PAGE

3. For annealing oligos, I use NEB buffer 2, in a PCR first at 95 C for 5 mins, then 70 C for 10 mins followed by slow cooling to room temperature (upto 20C) at the rate of 10 degrees per hour.

4. I do simultaneous digestion with Age1 and EcoRI

5. I get purify the digested vector with ethidium bromide staining and use a long-wave UV for not more than 25-35 secs

Can you guide me from here.

If you use unphosphorylated oligos, you could try ordering phosphorylated ends it does improve your chances of cloning.

Did you check the sequences to be sure that you dont get mismatched annealing. Your annealing time is rather long, I used a protocol that I found in a Promega brochure for cloning shRNA, 4ug of mixed oligos annealed 3 minutes at 90oC followed by 15 minutes at 37oC, then on ice, I diluted the mixture 1/ 10 for annealing.

I usually run the cut plasmid and oligo mixture on a gel to estimate the best ratio. Excessive amount of oligo leads to lots of problems and very low cloning efficiency. It has worked very well for me, with over 10 different Sh-RNA.

This is the buffer Promega recommended:

TRis pH 7.5

500mM NaCl

60mM MgCl2

10M DTT

Genevieve - if my method does not works then I will try yours. Thanks for the advice

hi Shivam,

You said that QC was performed by MS/PAGE, but how were the oligos purified? In my experience desalted oligos don't work nearly as well as purified oligos (HPLC or ideally PAGE)

I have also done my annealing in NEB buffer 2; your annealing conditions should be OK.

It may be helpful to try varying the ratio of oligo to cut vector for ligation

good luck!

Andrew - I dont know about that. we order them from SIGMA. these are the ones that are validated by them.

I would look into how the oligos were purified. If there were simply desalted, which is the default option when you order oligos, then in my opinion this is the most likely problem. Longer (e.g. >35 bases) oligos, such as those used for shRNAs, will be contaminated with a substantial fraction of truncated products as a byproduct of synthesis.

see

http://www.sigmaaldrich.com/life-science/custom-oligos/custom-dna/learning-center/best-purification.html

Shivam from what you describe I think you have most likely a structure problem with your particular set of oligos and the ligation is what fails. I've seen that before and would recommend that you use a ligation protocol which is called cycle ligation. Basically for 7-8 hours (not longer, it brings up the background of unspecific religation) have the ligation cycle between 10C for 30 seconds and 30C for 30 seconds. Afterwards 10 minutes 70C for inactivation.

There is a paper for that somewhere from another group, I can't recall who it was though. But this protocol has allowed me to overcome every ligation problem including - most problematic - intron structures when I was getting really desperate and other sequences with same enzymes etc worked fine.

Just be aware: the sensitivity is decreased with that protocol so you will get usually a significant amount of "empty" clones even if the cut sites don't fit.

Thanks Ulrike. I will try that as well. Btw, is this the paper for cycle ligation - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC145669/

Hi,

I have never worked with shRNA so I can't give you specific and detailed advice, but my understanding is that with different sequences cloning worked. You might try clone again one the RNA that worked as a positive control. lf that does not work anymore then you know you have a with reagents or protocol and focus your trouble shooting. Also, do you digest the phosphates on your vector to avoid it religates without incorporating your shRNA?

Aleardo - I normally dont do phosphatase treatment of the vector as its it digested with EcoRI and AgeI, which produce incompatible ends wrt to each other

Dear Shivam,

I don't know if you are still busy with the problem or not. If you want to clone shRNA in pLKO.1, you should order the TRC pLKO.1 from addgene and swap the shRNA sequence with a stuffer around 2 kb in the vector. If you have got validated shRNA vectors from sigma and try to swap another shRNA with the existing shRNA in the vector, it is not almost possible because the restriction sites have been manipulated by the company that you are not able to replace another shRNA.

Hi, I have the same problem, I am using pLko-tet on vector. Length of my SHRNA is 58bp. I ordered desalted oligos and I am not able to anneal or ligate them. Any help appreciated. I am specifically confused about which ratio to use for annealed oligos with double digested vector. I think in the annealed oligos mixture more than 50% are not annealled. 

Hi, where do you order 57 or 58bp oligo DNA for tet on plko shRNA construct? Do you order from IDT? I try to order 57bp oligo for tet on pLKO shRNA from IDT and I get this information "Warning: Due to the length of this sequence, this oligo will be processed with a Complexity Service. A reduced yield and purity guarantee will apply.

Warning: Due to the length and complexity of this oligo, a Level II set-up fee and reduced yield and purity guarantees will apply. This oligo will also require additional review from IDT Customer Care. If the result of this review causes any change in pricing from what is displayed in your cart, a representative will notify you prior to processing.". Does anyone get similar response from IDT? This seems that the 57bp oligo for shRNA contains specific structure and needs to be synthesized specially??? where should I order oligos?  Thanks.

In regards to my previous problem, I solved it and now it works everytime. I don't even need to sequence it to confirm. In my negative control I never got a single colony while in shRNA cloning I got like 20~50 colonies all the time. USE STBL3 BACTERIA ONLY.

Anyone who has the same problem please USE THIS PROTOCOL:

I attached the protocol, but in case if you can't see or download > go to the addgene pLKO.TET ON page download the protocol from there. Actually there are many protocol, but use the one with Bmi1 shRNA example.  FOLLOW THE PROTOCOL WORD TO WORD. It works like a charm. 

Here is the title and other details of the protocol.

The “all-in-one” system for the inducible expression of shRNA

Dmitri Wiederschain, Ph.D.

Novartis Developmental and Molecular Pathways, Cambridge, MA, USA

[email protected]

(617) 871-3616

Hi Shivam,

I have the exactly same problem now. Could you solve it and how may I ask? I found out my oligos are not annealed. It would be super if you can write to me!

Happy clonings. :)