PaigeSept2011

toc =September 2011=

September 5 - 9
Pre-Lab Questions: Set 1 1. How many microliters are there in 2.0438 milliliters? 2043.8µ 2. Should you ever use an air displacement pipette without a tip attached? Never use a pipette without a tip, you will contaminate the instrumetnt Pre-Lab : Set 2 1. 5' TACCGATCGTTTCCT 3' 5' CTTCTTGAAAAGTGA 3' 5' CCTCAACAGGGTTAA 3' 5' GAACAACTTAATCTA 3' 2. DNA strands would you get after 35 cycles of PCR amplification after starting with one strand? 2^35 = 34,359,738,370 3. Name two possible reasons for plateauing. - the number of original primers decreases as the number of new DNA severely increases - The original strands of DNA become weaker and less susceptible to the lessing primers Pre-Lab : Set 3 1. 3' CTACCGTATGCCTG 5' Pre-Lab : Set 4 1. Which type of organism would be easiest to transform: bacteria, plants, mice, or humans? - Considering some bacteria are single-celled I would think that transforming bacteria would be the easiest and then plants, mice and humans
 * September 6, 2011 **

**September 12-16**
**September 14, 2011** Today we met with April and Francis at Jordan and discussed our lab. We went over the steps of the GFP lab and we will be using Jelly Fish DNA for our lab. <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">__PCR: Polymerase Chain Reaction__ <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">__Ligation__ <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">The second step in our GFP cloning is ligation which is taking linear GFP and adding a cut vector to create a plasmid with GFP DNA inserted <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">__Transformation__ <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">__Plating and Selection__ <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Take a colony off the plate and see if it contains our GFP DNA <span style="font-family: Arial,Helvetica,sans-serif;"> We also went over the difference between blunt and sticky end enzymes. A blunt enzyme is when the nucleotides are cut even and sticky ends are when they are cut unevenly so there are loose ends for other dNTP's to attach too. Today was mostly review of the lab and explanations in what we will be doing for next week. <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">**September 16, 2011** <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Today we met and worked on using microsoft excel and it's different mathmatical and programing features. Below is an attachment of the work we did in excel for today. We also finished installing the MATLAB data to use for next week and recieved a packet in which we will be using for the program. We were asked to read Chapter 1 for next Friday. <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">The first step in PCR is the breaking of the H+ bonds of dsDNA which occurs at about 95 C
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Anneling of Primers is second. A primer is a short strand of single DNA designed to bind ends of DNA of interest.
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">There are forward and reverse primers going from 5' to 3' - it occurs between 50 - 60 C
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Extentions (Elongation) of DNA - 70 C We also learned about dNTP
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">free floating nucleotides DNA template = parent strand Taq Polymerase: thermal stable polymerase that withstand high temperatures for our PCR
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;"> --GFP-- + cut vector = GFP plasmid
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">insert plasmid into bacteria
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">heat shock

** September 19-23 **
<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">**September 19, 2011** <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Today we began the lab. We started with PCR: <span style="color: #001bff; font-family: Arial,Helvetica,sans-serif; font-size: 90%;">//PCR: Polymerase Chain Reaction// <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">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 <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">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. <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">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 <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">**September 21, 2011** <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">// PCR: making a gel using our PCR product // <span style="color: #10ca0c; font-family: Arial,Helvetica,sans-serif; font-size: 90%;">//Gel Electropharesis://
 * ~ <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Reagent ||~ <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Amount ||
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">5x PCR Buffer || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">4.0µL ||
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">DBTPs || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">5.0µL ||
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">GFP For. || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">3.0µL ||
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">GFP Rev. || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">3.0µL ||
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">DNA Template || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">4.0µL ||
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Taq Polymerase || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">1.0µL ||
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">**Total** || <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">**20.0µL** ||
 * 1) <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">primers - forward - reverse
 * 2) <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">jellyfish DNA
 * 3) <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">PCR buffer: medium for evreything to work
 * 4) <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Taq Polymerase: enzyme
 * 5) <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">dNTP : free nucleotides
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">separate DNA fragments through a gel material based on size of fragment. ethidium bromide
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">interculate:into major groove of DNA (carcinogen) GFP = 1 Kb target should be 1 Kb
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">1 fragment at 1 Kb Buffer is added to the gel to make it neutral
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">agarose was our buffer 2µL ofbuffer + 10µL PCR = our product for the gel
 * <span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">the more agarose the denser the solution for DNA to get through - higher gel --> greater resolution - glycerol: thick sugar enzyme

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 90%;">Today we put our PCR product into a gel to see if it had worked; and sadly it failed. No DNA was presented in our gel which we figured by using UV lights tosee our results. All that was present wasdye.We prepared 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.

__ **September 24-28** __
To<range type="comment" id="517243">day we used a nano-dropper to test our PCR products to see if any DNA template was present in any of our reagent due to the fact that last week we found out that our PCR didn't work by testing it in a gel. The reason our PCR didn't work we found out was because there was no DNA template in the tube we were using to begin with. This is part of our troubleshooting. We will next try to find a way to get our PCR to work so that we can finish the lab.
 * September 24, 2011**

This is the first part of our ligation notes:

This is a picture of part of our ligation notes we took today.