RobWardenWNB:M4D4

Biomaterials Engineering, Day 4, 5/9/07

Screen 2 Results

Sample	pCT-CON	pAu1	A	B	C	D
Num Colonies	6	1264	10	7	8	15

We had very low colony counts for our gold binders, which was expected. They were taken off of the plate that budded off after vortexing, so many yeast could have still been bound to the gold surface. We have chosen to sequence A and C, since A looked the best under the microscope, and C was chosen randomly due to little difference seen in between B, C, and D. A and C will be samples 13 and 14 for the class, respectively.

Sequencing PCR

Purpose

To create enough DNA that a sequencer can determine the sequence of our binding motifs.

Protocol

1. 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. Repeat with the second candidate you would like to sequence.
2. Close the caps and microwave the tubes in an eppendorf rack for 15 seconds. Next, move 5 μl of the microwaved mix, yeast debris and all, into a 200 μl PCR tube that you will be given.
3. Add 1 μL of each primer and 20 μl of PCR cocktail and 23 μL H2O 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.
4. Cycle the reactions as:
   1. 94° 4 minutes
   2. 94° 1 minute
   3. 52° 1 minute
   4. 72° 2 minute
   5. repeat steps 2-4 35 times
   6. 72° 10 minutes
   7. 4° forever

Summary

Today we chose samples to sequence by considering results of the second screen. These samples (A and C) were then prepared for sequencing by amplifying the variable region in a PCR experiment.