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How to anneal shRNA target together? - How to anneal shRNA target together (May/05/2005 )

Help!
I do not get my shRNA to ligate into my retro vector. The digest works because I see the vector being cut, and the transformation step works too as seen by a positive control. I think it's the ligation of the shRNA into the vector which might not work. Can somebody please tell me how they do it or give any good suggestions? Thanks!

-Shreyasi-

Hi Shreyasi,

Right now I am doing shRNA too, but not with retro vector. Very important, check every step before going to the next step.

1) Make sure your synthetic oligos are almost all in full length and get them from reliable sources
2) After annealing, run a gel along with ss-oligos, ds- and ss-oligos migrate differently
3) Make sure your ligase and its buffer work. After ligation, run a gel again to see if the insert is incorporated to the vector.

Hope that helps.

PS, i am cutting my plasmid and wait see how it goes.

-pcrman-

Hi there,
I did your suggestions and it it the annealing step that is not working. Do you also put your oligos at 90C and then allow them to go to room temperature?
Thanks!



QUOTE (pcrman @ May 5 2005, 05:39 PM)
Hi Shreyasi,

Right now I am doing shRNA too, but not with retro vector. Very important, check every step before going to the next step.

1) Make sure your synthetic oligos are almost all in full length and get them from reliable sources
2) After annealing, run a gel along with ss-oligos, ds- and ss-oligos migrate differently
3) Make sure your ligase and its buffer work. After ligation, run a gel again to see if the insert is incorporated to the vector.

Hope that helps.

PS, i am cutting my plasmid and wait see how it goes.

-Shreyasi-

Hi Shreyasi,

I heat water in a big beaker to 90C and put the annealing reaction tubes on top. Let the water to cool by itself to 37C (it may take 30-60 min) and then transfer the tubes to a heat block at 37C for additional 30-60 min.

Here is an example of annealed vs ss-oligo
Attached Image

-pcrman-

Thanks!!! I will try this!! This was very helpful!!! smile.gif


QUOTE (pcrman @ May 6 2005, 10:49 AM)
Hi Shreyasi,

I heat water in a big beaker to 90C and put the annealing reaction tubes on top. Let the water to cool by itself to 37C (it may take 30-60 min) and then transfer the tubes to a heat block at 37C for additional 30-60 min. 

Here is an example of annealed vs ss-oligo
[attachment=24:attachment]

-Shreyasi-

hi
i annneal oligos in ste buffer :
10X STE buffer
tris HCL ph 7,5 to 8 100mM
nacl 500mM
edta 10mM
boil them 95° 5' and let them cool on the bench
then check the reaction by agarose gel.

good luck

-fred_33-

QUOTE (Shreyasi @ May 5 2005, 05:13 PM)
Help!
I do not get my shRNA to ligate into my retro vector. The digest works because I see the vector being cut, and the transformation step works too as seen by a positive control. I think it's the ligation of the shRNA into the vector which might not work. Can somebody please tell me how they do it or give any good suggestions? Thanks!

Hi Guy,

I can not anneal my ss oligos to ds oligo, and I don't know why. After reached me, I diluted these ss oligos in the TE buffer at 100mM respectively, and then annealled in the STE buffer( reaction concentration :10 mM Tris-Cl,100MM NaCl and 1mM EDTA?. However when I checked them on 3% gel, I just saw the the bands, the size is same to the ss oligo.
Looking forward to your helps.

Max

-chiangmax-

hi
well i think your NaCl concentration is too high for a proper annealing.
Tip for anneal :
i heat a thermo block to 95° boil my oligos in it for 5min and then just switch it off, waiting the block to cool til RT. It is a longer procedure, but anneal is good.

-fred_33-