WEEK 5 ATTACHMENT SHARING

Heya guys……zahirah here….i’m currently attached to clinical pharmacology lab with michelle…if u guys think that we are in clinical lab..then u guys are super duper wrong..hahaha…well we are actually in a research lab doing studies on the genes of different ethnic group and see how it might affect the pharmacodynamics and kinetics of the treatment…think BPHARM….but then our lab deals with the major techniques in MBIO which is PCR and DNA sequencing…since we are together and to avoid u guys from reading the same thing twice….we will try our best to post up on different things….:)

Title: Running Gel electrohphoresis
Purpose: To determine the optimal temperature of the primers that we are going to use for the research by looking at the number and intensity of the bands.

REFER TO MICHELLE’S BLOG (http://jammys-bms.blogspot.com/) FIRST FOR THE OPTIMIZATION OF PRIMER

Before we run the gel…of course we need 2 make the gel first and it is prepared while waiting for the PCR to finish..save time a bit….soo..here’s the ‘ingredients’ and ‘recipe’ for the gel

For making agarose gel
Materials:
Agarose powder 1X TAE buffer Ethidium bromide Combs Tray
Conical flask Measuring cylinder

Method:
1.Prepare the tray by covering the open ends and pour DI water to measure how
much volume of TAE buffer is needed
-the volume needed is dependant on how thick you want your agarose to be
2.Pour away the DI water and place the tray in the fume hood
3.Calculate the amount of agarose powder needed
-this is calculated using the formula:
% of agarose gel x vol. of TAE buffer = weight of agarose powder
-since in this lab we use 1.5% agarose and the volume of TAE buffer needed is
70ml
thus: 1.5% x 70ml = 1.05g
4.Weigh 1.05g of agarose powder and put it in the flask
5.Measure 70ml of 1X TAE buffer
6.Pour the 1X TAE buffer into the flask and swirl gently
7.Place it in the microwave for 50 sec
8.Once you get a clear solution, bring the flask to the fume hood and swirl for a
few seconds first
9.Add 3.5ul of ethidium bromide into the flask after the solution has cool down a
bit
10.Calculation for vol. of ethidium bromide needed:
Stock conc: 10 000ug/ml
Dilution conc needed: 0.5ug/ml
M1V1=M2V2
0.5ug/ml x 70ml = 10 000ug/ml x V2
V=0.0035ml = 3.5ul
11.Swirl the flask gently to obtained homogeneity
12.Pour the solution into the tray and place the combs

Note: the volume of 1x TAE buffer, agarose powder and ethidium bromide and the number of combs needed is dependant on the size of the tray/agar. For this one, it is for a normal size agar that is able to hold two combs. The thickness of the comb is also dependant on what product we are going to run. For primers, we use a comb with big and thick wells as we are going to load all the PCR products.
For running gel electrophoresis
Materials:
6x loading dye PCR products 100bp ladder 1.5% agarose gel

Method:
1.Place the agarose gel into the gel electrophoresis machine
-ensure there’s enough TAE buffer to cover the gel
-the wells is placed at the negative part of the machine
2.Pipette 2ul of 6x loading dye into each PCR tubes and mix
3.Load the samples to each corresponding well(starting from the 2nd well)
Eg. 1st PCR tube-to the 2nd well
12th PCR tube- to the 13th well
-must ensure that we don’t jumble up the tubes and the loading part because
each tubes represent the product that has undergone different annealing
temperature
4.Pipette 2ul of 100bp ladder to the 1st and last well( after the 13th well)
5.Run the gel at 120V for 15-20 min

++Initially we want to post up the pictures we obtained after gel electrophoresis but when we ask our supervisor for permission, he did not give us a clear cut yes….soo sories guys…but u can refer to your MBIO lecture notes the topic after cloning…the bands we obtained are almost similar to that…

A BIT ON LMQA...
All staff is required to attend safety modules which even include a module on centrifuge safety..at the end of each module, we actually have to sit for a test and even do assignment. All the work done in our lab are pretty manual..except for those that must be done by machines like DNA sequencing, etc...not that advance like nisha's where there's even an automatic pipette filler..our fridge actually have warning that says may contain biological agent because we do store blood from patient's in the same fridge we store our reagents...and there are some labs that have the radioactive symbol(the three triangles) because that particular lab deals with radiation/radioactive materials or they used to deal with it.....

so my posting is till here......shall continue the nxt time i post....wishing everybody all the best for your SIP/MP and enjoy....tata...:)