4 replies to this topic

[speci]

Dear All,

I'm actually having problems to clone a 95 bp fragment into a 7kbp vector.
I ordered the dsDNA 95 bp oligo, containing a mitochondrial targeting sequence (MTS)  from microsynth.

I included the restriction sites for EcoRI and BamHI, with additional 6 bp facing outwards.

Sequence:
tctgcagaattcATGTCCGTCCTGACGCCGCTGCTGCTGCGGGGCTTGACAGGCTCGGCCCGGCGGCTCCCAGTGCCGCGCGCCAAGggatccaccatg

Upper letters: MTS
Underlined: Restriction sites

I Digest 5ug of the oligo with both High fidelity enzymes together (NEB buffer 4 100% activity) for 2hrs at 37°C. Afterwards I run a 2% agarose gel and purifiy the band.

I do the same for the vector. I don't dephosphorylate the vector, because i didn't phosphorylate the ordered oligo to avoid contactamer formation. Control ligation without Insert gave no or only very few colonies.

In the next step I measure the concentration after purification.

For the ligation I use 50 ng of Vector. I tried several Vector:Insert ratios (molar). Ranging from 1:3 to 1:20.
I perform overnight ligation with the T4 Ligase (Invitrogen) at 16°C.

I used XL1-Blue for transformation on freshly prepared agar plates containing Amp.

This gave me approx. 15 colonies on some plates. No or very few on control plate without insert.
I did control digest with EcoRI and BamHI. Only one clone showed a band at approx. 100 bp. I sent this clone for sequencing. Surprisingly there was a 65 bp insert between the two restriction sites which had nothing to do with my MTS sequence .
Do you have any suggestions? I think there is a problem with the digest of the short oligo....

I know that it would have been better to order sense and antisense ssDNA and perform annealing which results directly in the correct sticky ends for ligation. But do you have any suggestions what I could try with my ordered oligos. I don't want to trash the 100 bp oligo...

Looking forward to your answers!!

Thanks in advance

speci

[bob1]

Did you get the oligo as a HPLC or PAGE purified form - if not, you probably have the majority of the DNA in the tube not being the full sequence, which will mean that the cloning will be very inefficient.  Did you order it as a dsDNA or did you do the annealing yourself?

Dephos of the vector isn't necessary as you have non-complementary ends.  The insert doesn't need to be phosphorylated either as you are digesting it, which would give you the correct phospho ends.

Did you perform a control ligation to ensure that the ligation was working?

I recently did the ssDNA annealing like you mentioned very successfully, using a 15:1 insert:vector molar ratio and less than 5 ng of vector.  The manual I was following (even though I wasn't using the kit it came with, these were custom DNA sequences) was the Invitrogen BLOCK-it Pol II miR RNAi expression kits manual, which contains some useful tips.

[speci]

I ordered the oligo PAGE purified, so this shouldn't be a problem.
Ah Shit...I did a stupid mistake...

I ordered only the oligonucleotided which i posted above...
This is only single stranded...so embarassing...

If I order two oligos and anneal them. Can I perfom this in water? I don't want to have salts etc. for further cloning steps.

Edited by speci, 28 April 2013 - 06:39 AM.

[bob1]

You can't do the annealing in water, you need some salt in there.  Apparently you can do the annealing in 1x ligase buffer (though the ATP may need to be supplemented for ligation).  My recipe for a 10x annealing is 100 mM tris pH 7.5-8, 500 mM NaCl, 10 mM EDTA.  Check out the manual I suggested, it has really good information in it.

[speci]

Thanks for your reply, I performed the annealing today in your proposed annealing buffer. Let's see how it works!

Thanks for your help!

Best wishes