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Appendix A
Techniques

A.1  Nucleic Acid Determination by Spectrophotometry

Values found outside these acceptable ranges are colored red in the text of this notebook.

A.2  Pipetting

A.2.1  Multichannel pipetting

Multichannel pipetting brings a little sanity to the montonous life of a middle throughput experimentalist. Here are a few tips that I've figured out or picked up from other people along the way.

Are you having problems sucking the same amount of sample from each channel?

I think this is a never ending problem and the most frequent one. But it doesn't have to be there if you do a little work. I assume your pipettor is calibrated, so the error is your own fault not the instruments. If it's not calibrated once a year, it should be.
When I first started I had a tendency to just pipette anyways if the differences were small between because I thought they would average out in the end and I was lazy. This is a bad approach. It is usually the same channel having the same error because there isn't a good seal, and the seal often gets worse all of this could lead to some nice correlations in your figures that you spend years exploring as scientifically interesting results that can only repeated with your pipettor.
The easiest way to get a big boost in accuracy is to buy the tips recommended by the manufacturer. These are usually the most expensive tips available for your pipettor, and they are typically made by the manufacturer of your pipettor. The second easiest way, but one the requires a little work once a week or so is silicon grease. Adding a little (and I stress little) amount of this magic goopy stuff to each channel allows the channels to form a much better seal with the tip, so when you set the pipetter to suck up 10 ml you suck 10 ml with each channel rather than a random number between 5 and 10 ml in each and the rest air.
This trick was taught to me by Jamey Wierzbowski. I'm not sure how he does it but here's how I do it:
  1. dip a Kim wipe into a container of silicon grease trying to get a small amount on it
  2. rub the kim wipe / grease across each side of the channels coating them well but not globbing it on there; avoid getting it onto the holes where the air is aspirated from
  3. use your fingers (with a glove on of course) to rub the grease already on the channels into those hard to reach places between the channels (if you have giant fingers you should probably figure out a different way to do this
  4. wipe of the channels with kim wipes until you can no longer see any grease on the channels
  5. pipette some water with all 12 channels and if when you eject the tips they don't shoot off or shoot off very hesitantly, you have way too much grease on there. wipe more off with kim wipes and be through this time you lazy bum.
  6. You are now ready for pipetting heaven. Pipette and enjoy seeing exactly the same amount of liquid in each tip.




Wed Feb 15 11:16:18 EST 2006
On the last 384-well plate of my 9000 qPCR reactions in the initial ChIP-Chip study, I made a discovery that is more important than the silicon grease. The most important factor is to have the tips made by the same manufacturer as the pipettor. I was using generic tips from Fisher that were supposed to fit the Finnipipette multichannel we have. They did kinda, but it is no comparison to the real Finntips. The tips are more expensive then the generic ones however, by using refill pages the price is almost exactly the same (one-hundredth of a penny more per tip) and I can save the environment (FINNTIP 10 REFILL KIT Fisher Part # 14-245-149, FINNTIP 10 REFILL Starter 14-245-148). With the starter you get 4 tip holders, so it is probably necessary to buy 2-3 of those.