September+19-23

Today we began the lab. We started with PCR //PCR: Polymerase Chain Reaction// We began by practicing our pipetting skills by using a 20µL pipette and began with filling it with water first by doing 10µL and then 5µL and then even smaller 2µL so we could get a feel with how the much a certain amount of liquid should look in the pipette once we attain it. Then we began the lab. We added the reagents in this particular order But for one tube, instead of adding 1.0‍µL to the tube we added 3.0 instead and we will see how that affected our results on Wednesday. After filling our tubes we set them in our PCR machine in which we left to complete 35 cycles for Wednesday in which we can continue on to the next step: Ligation. We also did an acid and base lab; we took a phenol red and added an acid to it which turned the solution from yellow to a pink colour. Then we went and added more base until it returned to yellow again with a few flicks.
 * September 19, 2011**
 * ~ Reagent ||~ Amount ||
 * 5x PCR Buffer || 4.0µL ||
 * DBTPs || 5.0µL ||
 * GFP For. || 3.0µL ||
 * GFP Rev. || 3.0µL ||
 * DNA Template || 4.0µL ||
 * Taq Polymerase || 1.0µL ||
 * **Total** || **20.0µL** ||

And following are the Pre-Lab questions for what we covered in the lab today: Pre-Lab Questions: ‍ 1. How many microliters are there in 2.0438 milliliters? 2043.8µL 2. Should you ever use an air displacement pipette without a tip attached? Never use a pipette without a tip, you will contaminate the instrument

**September 21, 2011** // PCR: making a gel using our PCR product // 1. primers - forward - reverse 2. jellyfish DNA 3. PCR buffer: medium for evreything to work 4. Taq Polymerase: enzyme 5. dNTP : free nucleotides

//Gel Electropharesis:// - separate DNA fragments through a gel material based on size of fragment. ethidium bromide - interculate:into major groove of DNA (carcinogen) GFP = 1 Kb target should be 1 Kb - 1 fragmetn at 1 Kb Buffer is added to the gel to make it neutral - agarose was our buffer - the more agarose the denser the solution for DNA to get through - higher gel --> greater resolution - glycerol: thick sugar enzyme

2µL ofbuffer + 10µL PCR = our product for the gel Today we put our PCR product into a gel to see if it had worked; and sadlyitfailed.No DNA was presented in our gel which we figured by using UV lights tosee our results.All that was present wasdye.Weprepared our gel and we ran it from the positive to the negative side since DNA is negative.We filled the tray with buffer solution untilit was filled until the comb. Then once the solution became solid we pulled the comb out and it created wells for our product to go into.We then put 12µL of our PCR into the wells and ran then gel.