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ds-oligo cloning/ligation issues


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18 replies to this topic

#16 Trof

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Posted 27 February 2012 - 05:19 AM

I see. The uncut vector and re-ligation is the main problem.

But I don't really like PCR much when talking about several kbs. If it was just ordinary bacterial plasmid, that selects out mutants in required genes needed for selection, then it may not matter. But my plasmid is a mammalian expression vector and I can't have it hit by some mutation in PCR, because it could then have lower expression that wild type for example, that would screw the subsequent experiments. The probabilty of this is low, but I would have difficulty to find it out.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

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#17 phage434

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Posted 27 February 2012 - 06:16 AM

Yes, that could be an issue. Use of high fidelity enzymes, especially Phusion, mitigates this, but if you really need to be certain, there is no substitute for sequencing your final version.

#18 Trof

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Posted 30 March 2012 - 11:57 AM

I see what you meant by the background. I didn't purify neither the insert nor the vector and made 3:1 and 5:1 ratios. Got pretty low colony count (around 30 on plate with 200 ul), only one of thirty tested colonies was positive for the insert, and that one had wrong size on the restriction. All others were empty.

I'm trying again on Sunday, gel purify both insert and vector and ligate in the ligation buffer this time, see if this approach is better.

Anyway I learned by this that I can have false positives on colony PCR from the remains of ligation reaction, so I guess it wasn't completely lost time.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#19 Trof

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Posted 03 April 2012 - 07:38 AM

Now, I digested old and new vector (Fast digest enzymes, 10 minutes), run them on gel, cut off insert (3,3kb) and linearised second vector (7,1kb) (used long wave UVA, tried to be quick as possible), isolated by Qiagen kit (MinElute for inserts and Qiaquick for vector, it's up to 10kb), measured concentration.
Made 5:1 molar ratio only, because the overal amount I got from gel was not enough (around 10 ng/ul), so I had 30ng of vector and 69 ng of insert.
Ligated 1 hour at 22 (Fermentas manual states 10 min is enough, but 1 hour can increase number, no PEG in buffer), and transformed XL1-Blue subcloning grade cells, that has been thawed once with 5ul of 20ul ligation reaction.
I got average 2 colonies/plate, most negative on colony PCR and the only possitive showed no insert on RE.
After that I remembered I forgot to heat kill the ligase so I repeated the transfromation next day with ligase inactivated, but numbers were similar, two PCR possitive clones has to be checked tommorow.

I'm trying to find the bottleneck. I will digest more DNA now, to have at least for 1:1 and 3:1 ratios using 50ng of vector, I will do single-cut-vector and single-cut-vector+ligase control. Also I used Fast AP to dephoshorylate vector right during the restriction, which was admissible in the manual, but now I will do it after digestion, heat kill.
But the main problem is getting low number of transformants. Bacteria are AFAIK fine, I'm using same for a month now, same transformation procedure. But I'm considering using those from non-thawed tube, that should increase the efficiency. On the other hand there are only two left (handy 500ul packing Posted Image ) and I have three inserts (same size, different mutation) and the difference shouldn't be such high.

Is there anythyng else I can do different? I can hardly do something with the UV step, but I think 400 - 315 nm shouldn't cause considerable problems, or would the potential damage be compensated by higher amount of DNA in ligation reaction, like 100 ng of vector and appropriate amounts of insert? Ligase and enzymes are all new so they should work, restriction seems fine judging by the bands of first vector with insert. The concentration ig gel excised fragments could be checked on gel, however this time the concentration was too low to be seen, for same reason there is probably no point in putting the rest of ligation on gel.

Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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