Bryan Hernandez/20.109/Lab notebook/Module 4/Day 4
PCR of gold binding candidates
--Bryanh 13:32, 9 May 2007 (EDT)
purpose: Prepare DNA via PCR of gold binding candidates from the library to send for sequencing.
- Begin by counting the colonies that arose from the gold binding experiment you performed last time. The two library candidates that seem to have the highest affinity for gold will be sequenced.
- You will use the microwave to release the DNA from the yeast. On the tip of a sterile toothpick, pick-up a dab of the correct colony from your Petri dish and swirl it in 20 μl of sterile water in an eppendorf tube. Be sure to label the tube so you know it belongs to your group and which candidate it contains. Repeat with the second candidate you would like to sequence.
- Close the caps and microwave the tubes in an eppendorf rack for 15 seconds. If you haven’t been wearing gloves, start here. PCR is a sensitive technique and trace amounts of DNA from your fingertips can be detected. Before you proceed, clean up a bit, e.g. wash the barrels of your pipetmen with a paper towel and some 70% EtOH. You could also wash your bench area. Next, move 5 μl of the microwaved mix, yeast debris and all, into a 200 μl PCR tube that you will be given. Again label your tube well (write small!).
- Add 45 μl of PCR cocktail to each PCR tube and leave the tubes on ice until everyone is ready. The recipe and template sequences are listed at the end of today’s protocol.
- Cycle the reactions as:
- 94° 4 minutes
- 94° 1 minute
- 52° 1 minute
- 72° 2 minute
- repeat steps 2-4 35 times
- 72° 10 minutes
- 4° forever
While these reactions are cycling, you and your partner should work on the research proposal that you will present to the class next time (see FNT, below).
If we had more time today we would confirm that there is product in your reactions and we might even measure its concentration and remove the PCR salts and buffers before sending it off to the sequencing facility. Since there is insufficient time, we will blindly send it off and hope for the best. Sequencing reactions require 100-200 ng of DNA, and 6.4 pmoles of sequencing primer in a final volume of 24 μl. For each PCR product, make a 1:10 dilution in water and aliquot 2 μl to a full-sized eppendorf tube, labeled properly. A mixture of water and sequencing primer will be added (22μl) and the samples will be taken to the Biopolymer facility in E17 for sequencing. The data will be available for you to examine one week from today. Keep your fingers crossed.
colony counts for library rescreens:
Lib B: 8 (labeled as sample 5)
Lib C: 25 (labeled as sample 6)
summary and interpretation
Our library rescreening results show that our Lib C colony is likely a gold binder because of the high colony count as compared to PCT-CON. In comparison to the pAu1, however, it is very poor. This might mean that it is simple less effective at binding gold or there were errors in our procedure. Sequencing will allow us to see how different these sequences are and then we can say more. Lib B was scarcely higher than PCT-CON. I am not convinced that this is a gold binder. If we had time, I would like to do another binding experiment with Lib B to check to see if it was a non-specific binder. It could simply be very bad at binding to gold though. word.