Sunday, July 29, 2007

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 machin
e
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...:)

8 comments:

VASTYJ said...

hi zahirah!

why do u have to put the flask in the fume hood when pouring the agarose powder? is it because the powder will irritate your nose or it is toxic?
hope u're enjoying SIP!

Ying Ying
TG01

royal physicians said...

Hey zoRRo,

u know what, I also run gel but rarely, and after reading your post then i know why i add 3ul of EtBr, haha..i tot it was a standard thingy..lol

Aniway, just would like to correct the unit used for the concentration, it should be ug/ml instead of ug...concentration rite...:))

Nisha
TG02

we are the XiaoBianTai-7! said...

Hey

What is ethidium bromide for?

Thanks!

Adrian TG01

royal physicians said...

Hey Zahirah

I'm not sure if you mentioned it in your blog but i was wondering what is the use of your TAE buffer and when you mentioned ethidium bromide, is it already mixed with your TAE buffer? Are there any special things to take note of when handling ethidium bromide? sorry for the many questions. hope to see you soon

Johanna TG02

royal physicians said...

TO: Ying Ying

heya girl, well we are not pouring the powder but more of the agarose solution (coz liquid form already after mixed with TAE buffer)...the reason why we do it in the fume hood is because:
1. ethidium bromide is a powerful mutagen and it may cause heritable genetic damage, thus it's best to do it in a designated area and have a protective shield so that in case the bottle spill or it splash, there's this glass 'window' to protect u..u noe the fume hood glass thingy..yah..:)
2. because we need to swirl the flask containing the agarose solution and ethidium bromide after it's added, there might be a chance that we might swirl too hard and that some of the solution splash out, so we need that protective glass
3. both the the agarose and ethidium bromide are harmful if swallowed or inhaled and they can cause irritation to the eyes and respiratory tract. as while we are doing this, we are not using goggles, we need to protect ourselves especially our eyes, and i dare not imagine what happens if the solution gets into the eye with the absence of eyewash in all the labs.washing the eyes in the sink would be soo uncomfortable.:)

royal physicians said...

TO: Nisha

hahahaha, i wish it's standard also so don't have to keep calculating....noted on the concentration part...i make the changes already...soo sorry coz my lab used to saying ug even for conc coz at the end of the day it'll be cancelled out...haha

royal physicians said...

TO: Adrian

Ethidium bromide is able to intercalate the DNA, thus allowing visualization of the DNA bands. This is because when we run the gel electrophoresis, the DNA, being negatively-charged, will move towards the positive site (anode). the ethidium bromide, on the other hand, will move towards the negative site (cathode). at some point, because they are moving in opposing direction, the ethidium bromide will intercalate (insert themselves in between the bases) and thus allowing us to visualize using the bioimager.

royal physicians said...

TO: Johanna

TAE buffer is the common choice of buffer used in gel electrophoresis to separate especially linear, double stranded DNA. As we are going to run the gel in TAE buffer, therefore, to dissolve the agarose powder, we also need to use TAE buffer. if we use two different buffer, it'll affect the running of the DNA. i think the DNA will just run 'wildly'. ha3.
the ethidium bromide is added separately. the agarose powder must dissolve first in the TAE buffer, then ethidium bromide will be added. this process of adding and then poruing the agarose solution is done in the fume hood and of course, lab coats and gloves are important. after casting the gel, the gloves must be immediately thrown away and the flask must first be 'flooded' with water for a few minutes(remember like washing the waste bottle for MCT)then washed with Decon90. oh yah, the pipette we use to pipette the ethidium bromide cannot dispense away the tip and thus we have to use our hands to remove it, and we have to be careful not to come into contact with it and remove it by the top of the tip...not the sharp end part..and also not to come into contact with the inner side of the cap for the ethidium bromide bottle.