Sunday, September 9, 2007

Lab Technique

Hiya...I shall talk about purification of primers and followed by DNA sequencing after finding the optimization temperature,a lab technique that I have done after PCR. The purpose of purification is to ensure that the unwanted unbound stuff like unbound primers or excess buffer will not be present and thus will not affect the results after DNA sequencing is done. Feel free to ask any questions ya..

Purification of PCR Products:

Materials

Shrimp Alkaline phosphotase (SAP)

Exonuclease 1 (Exo 1)

Method:

1) Prepare a master-mix containing both the enzymes for purification. (Refer to table below for purification reaction mixture set-up.)

Purification reaction mixture set-up

Components Required:

Volume Required/ul:

Master-mix Required/ul:

SAP

1.0

80

Exo 1

0.5

40


1.5

120

2) Pipette 15ul into 8 eppendof tubes. Using a multi-channel, pipette 1.5ul of the master-mix into the wells of the PCR plates.

3) Place the PCR plates into the machine.

DNA Sequencing (Cycle sequencing):

Materials:

Sequencing dye

Sequencing buffer

Forward primer

Reverse primer

Autoclaved water

Method:

1) Prepare a master-mix containing all the components for cycle sequencing. (Refer to table below for cycle sequencing reaction mixture set up.)


Cycle sequencing reaction mixture set up

Components required:

Volume Required/ul:

Master-mix Required/ul:

Sequencing dye

1.0

80.0

Sequencing buffer

0.5

40.0

Forward primer OR reverse primer

0.4

32.0

Autoclaved water

0.6

48.0


2.5

200

2) Pipette 2.5ul of the required reaction mixture into a new PCR plate.

3) Pipette 2.5ul of the correct purified PCR products into the new PCR plate, which contains the reaction mixture. Ensure it is mix well.

4) Spin down the new PCR plates.

5) Place the new PCR plates into the machine.

DNA Sequencing (After cycle sequencing):

Materials:

3M of Sodium acetate

125mM of EDTA

100% Ethanol

70% Ethanol

Formamide

Method:

1) Spin down the PCR products in the PCR plates.

2) Prepare a master-mix of the stock solutions (200ul of 3M sodium acetate and 200ul of 125mM of EDTA).

3) Pipette 2ul of master-mix of the stock solutions into each well of the PCR plate, using a multi-channel.

4) Dispense 25ul of 100% ethanol into each well of the PCR plat, using a multi-channel.

5) Pipette everything from the PCR plates and transfer them into sequencing plates, using a multi-channel.

6) Wrap the sequencing plates with aluminum foil and incubate them at room temperature for 15 minutes. Dispose the PCR plates.

7) Centrifuge the sequencing plates at 3000G at 4oC for 25 minutes.

8) Invert the sequencing plates using 3 pieces of tissue paper and pulse them at 200G at 40C for 20 seconds.

9) Dispense 70% ethanol into each well of the sequencing plates.

10) Centrifuge the sequencing plates at 1700G at 4oC for 15 minutes.

11) Invert the sequencing plates using 4 pieces of tissue paper and pulse them at 200G at 4oC for 20 seconds.

12) Repeat step 11, using the other sides of the tissue paper.

13) Place the sequencing plates into a vacuum dryer, not more than 7 minutes.

14) Pipette 20ul of formamide into each well of the sequencing plate/

15) Cover the sequencing plates with rubber mats and place the sequencing plates in the septa. Place them into the sequencer machine.

Michelle TG02 0503808H


4 comments:

Kent said...

Hi,

Would like to ask what is the fuction of the sodium acetate and EDTA in the PCR master mix, and what formaldehyde is used for in the sequencing reaction.

Thanks!
Kent
TG01

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

hiya kent,
the function of the sodium acetate and EDTA us to terminate the cycle sequencing process.
We used Hi-Di formamide which is a highly deionized formamide to dissole the DNA pellets before placing the sequecning plates into the sequencer machine.

michelle tg01 0503808H

Distinction in Disaster! said...

hey michelle,

what is the purpose of formamide at the last part of your DNA sequencing?
by the way, u all purify the PCR products by enzymes? i use the purification kit in my lab from the manufacturer, is there a difference in the principle?

AI Tee
0503160D

Distinction in Disaster! said...

sorry michelle, ignore my 1st quest.. i nv saw the reply u made after i post my comment..

Ai Tee