RE (MspI) Digestion of PCR Products

Griffith University Gold Coast supervisor at ipnet.ins.gu.edu.au
Sun Apr 16 00:07:01 EST 1995


Hello,

I Have been attempting to cut a 372bp PCR product with MspI, 
but I have not had much luck so far.

The PCR fragment in question contains a polymorphic MspI restriction
site, which has caused me problems in the area of not being able to
tell the difference between a homozygote for the restriction site
that is being incompletely cut or if it is in fact a heterozygote
for the restriction site. (i.e. I am yet to encounter a definite
homozygote for the restriction site!)

My first attempts consisted of just adding MspI and its' reaction
buffer to some straight PCR product (20ul). Excess MspI has been used
in each case (5U, 10U, 20U, even 40U) all with little success.

I have tried cleaning the PCR product with a chloroform-isoamylalcohol
(24:1) extraction followed by EtOH precipitation (with 1/10th Na Acetate).
And then digested with as above.

I have heard that it is not always necessary to clean up your PCR
product but MspI can often be tempermental in its' performance.

If there is anyone out there who could shed some light on my seemingless
fruitless MspI adventure, It would be greatly appreciated. 

Thanks (in advance).

ps. I'm visualizing via EtBr stained agarose gels (1.6%).

Dale.




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