Inquiry re: PCR setup and Pipet Accuracy.....

TIM CHIPMAN 901106c at axe.acadiau.ca
Fri Jul 9 13:57:12 EST 1993


I have an inquiry for all the PCR experts out there.

I have just started doing RAPD PCR using honey bee DNA, using RAPD's from 
UBC. I had sucessfully completed a few runs last week of up to 12 reactions 
at the same time, although I had found the reaction setup a bit hairy, since 
the volumes are quite small (12.5 ul per reac, total)and I am used to more 
"bucket chemistry" techniques...(ie: volumes of no less than 50 ul)

anyway. This week, I attempted to set up 41 reactions to go in the PCR. I 
mixed up a pre-mix slurry of TAQ, 10x incubation buffer, and dNTP slurry (a/
t/g/c). I then aliquoted this off into seperate groups, added various 
different primers (I attempted a total of 8 different primers), split these 
off, and then added various template DNA (I used a total of 8 different 
template DNA types). However, when aliquoting various things out after the 
initial pre-mix stage, I found that I was losing stuff along the way in a 
severe fashion. I lost completley enough premix for 5 reactions, and also 
had some others that came up looking suspiciously small/short on reagents.

My question is thus: 

How much "overshooting" is standard for a pre-mix. (ie: 41 reactions 
desired, mix up enough for 42? 45? 50? ...? I am keen to not overshoot too 
much, because TAQ is not exactly inexpensive.

Also: How large a margin of error is "acceptable" or expected when using 
eppendorf digital pipettes? We are using units with the ranges:

100-1000ul
10-100ul
2-10ul
0.5-10ul

Also: we just got a new shipment of yellow tips in from fisher, and they 
seem AWFUL at first glance... upon expulsion of the tip, it immediately 
seems to draw some liquid back up.... does anyone have any recommendations 
for what yellow tips they find the best?

Also: we have done simple cleaning procedures on the pipetters (unscrew 
shaft, gently clean plunger apparatus with kimwipes). Does anyone have any 
suggestions for additional cleaning or calibration procedures that would be 
of use...? Thismorning, I spent a while shuffling about some water, trying 
to ascertain how grim the situation was. (ie: use 10-100 ul unit, put 100ul 
in eppendorf, then use 2-10 unit to attempt to pull of 10x10ul...usually 
running out at about "80" ul....etc....etc....)

Thanks! I really appreciate all assistance that you may be able to give me!

Tim Chipman
901106c at axe.acadiau.ca
901106c at dragon.acadiau.ca



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