Ligation

[ChenHA]

Deitiker, Philip R wrote:
> I have repeated the ligation. 
> 
> 1. Positive control. Plasmid 1.6 ul/17 ul of cells.
> No ligation. No gel purification. 
> 
> 2. Positive control. BamH1 cut Plasmid
> no Insert, gel purified.
> 
> 3. Negative control. BamH1/EcoR1 purified by
> gel and ligated with no insert. 
> 
> 4. Test. BamH1/EcoR1 purified with added insert
> at 3:1 ratio. 
> 
> The vector DNA was visible on the gel without transillumination
> and was cut out with visible light. The remainder was examined
> by Transillumination, some loss of DNA most DNA however was
> in the band. 
> 
> All transformations at 1.8 ul/17 ul of cells, all plates
> are.  
> 
> Result. 
> 
> 1. ~4000 CFU per plate
> 2. ~250 CFU per plate
> 3. 13 CFU
> 4. 11, 13, 19 CFU per plate.
> 
> 3 and 4 are not statistically different.
> 
> Conclusion. 
> Problem is not primarily in  cells, either may have ligation reaction
> problems, gel cleanup problems, DNA solubility problems. 
> or Insert not digested properly. 
> 
> I think it is worthwhile to order primers and reamplify
> products. 

You can re-order the primers, but in the mean time you can also screen 
the colonies by PCR for inserts - pick colonies, disperse in 20ul water, 
use 10ul for PCR (20ul of PCR reaction is enough), add the rest to 1 ml 
of LB for use later if found to be positive.  Best to use 1 primer from 
vector and 1 primer from insert for PCR. Check result on gel.

It is very likely that there will be some plasmids with inserts - if 
there are single-cut DNA that can circularise, a good proportion may 
become ligated to the insert so they can't actually circularise (because 
they now have the wrong ends), therefore the actual background are lower 
than the control plate unless those colonies are all from completely 
uncut plasmid.


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