pcr setup,pipette accuracy

[john hachey]

>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.


I usually make up enough for 2-4 extra reactions to account for pipetting
 errors and to have enough left over for at least one reaction due to 
semi-inevitable spill/screwup.


>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


I have always assumed for any chemistry that +/- 5% is acceptable.


>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?

This sounds like a pippettor problem...a little grease applied to the o-ring
in the pipettor barrel should improve the seal.


>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....)

For pcr we use a set of autoclavable pipettors, but I would think the regular
kind could stand some soaking in soapy water followed by a rinse once in a 
while.
I think the most reliable way to calibrate your pipettors is to weigh the
water that it dispenses (1mg/ul).

>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