Sunday, July 29, 2007

Lab Technique

Hiya, I’m currently attached to clinical pharmacology lab doing research work. Most of my work involves PCR techniques and DNA sequencing. I’m currently doing my SIP and MP with Zahirah. Today, I’m going to share with you guys an experiment, which is important because it will affect our subsequent experiment results.

Experiment title: Finding the optimal temperature of primers.

PCR-Materials and Method

-It is good to cultivate a good practice to prepare a mastermix.

Reagents Required:

Volume Required/ul:

Mastermix Volume/ul:

Autoclaved water

6.4

89.6

Buffer

1.0

14.0

MgCl2

0.6

8.4

dNTP

0.3

4.2

Forward Primer

0.2

2.8

Reverse Primer

0.2

2.8

Test DNA Sample

1.0

14.0

Taq Polymerase

0.3

4.2


10.0

140

-Add all the required reagents into a large 1.5ml eppendof tube(This is your mastermix.), and pipette 10.0ul into each 0.2ml PCR tube. These reagents must be placed in a ice-box or chiller to prevent degradation of the components in the reagents, particularly Taq Polymerase. Taq Polymerase is to taken out only when it is required and it must be returned back into the fridge immediately. Ensure the mastermix is well-mixed by centrifuging and vortexing it for a few seconds.

-Place them into the PCR machine in one straight row because different columns have different temperature. We are performing gradient PCR in which we do not know the annealing temperature for the primer hence we have to program the annealing temperature range to be from 50C to 60C and the machine will set up a specific temperature for each different column. The PCR conditions are initial denaturation at 95C for 3 minutes, denaturation at 94C for 45 seconds, annealing at the temperature range being set for 45 seconds, extension at 72C for 45seconds and final extension at 72C for 7 minutes. The PCR conditions are repeated for 30-35 cycles to have sufficient amount of particular segment of the gene that is required.

-After the PCR process is done, we must perform gel electrophoresis and operating the Bio-imager to view the DNA image. After analyzing the DNA image (Thickest band.), we must check the specific temperature of the column which the thickest band is seen from. This temperature will be the optimal temperature of the primer.

Refer to Zahirah’s posting regarding on gel electrophoresis as she will explain it in details. Hope you have learnt something from my SIP and feel free to ask any questions ya.

Posted by: Michelle TG02

2 comments:

Distinction in Disaster! said...

Hello michelle!
Just 1 question:
So gradient PCR is just used to find out the optimal annealing temperature?

Thanks!

Charmaine Tan
TG01

J.A.M.M.Y.S said...

Hiya charmaine,
yeap,gradient PCR is to find the annealing temperature for the primer. It is called gradient PCR because we set a range of temperature(eg.55-65C) and the machine will auto set a specific temperature for each column. :)
michelle