February 14, 2018

Standard PCR reaction:

DNA template (50 µg/µl) 2 µl
Forward primer (10 µM) 1 µl
Reverse primer (10 µM) 1 µl
dNTPs (5 µM) 2 µl
10x Taq reaction buffer 5 µl
Taq DNA polymerase 0.5 µl
ddH2O 38.5 µl
total volume 50 µl

PCR master mix:

DNA template (50 µg/µl) 2 µl
Forward primer (10 µM) 1 µl
Reverse primer (10 µM) 1 µl
2x PCR master mix 25 µl
ddH2O 21 µl
total volume 50 µl

Our study PCR master mix:

DNA template 4 µl
Forward primer (10 µM) 0.5 µl
Reverse primer (10 µM) 0.5 µl
2x Ampli Gold Taq master mix 12.5 µl
ddH2O 7.5 µl
total volume 25 µl
PCR program:
Initial denaturation (active Taq polymerase)
Denature
Annealing Program
Extension
Final Extension (Give sometime for enzyme to fill the gaps)

PCR machine setting:

1. 95oc 5 mins
o
2. 95 c 20 sec
o
3. 60 c 20 sec
o
4. 72 c 30 sec (20-25 bp of PCR product/sec)
5. 40 cycles
6. 72oc 5 mins
o o
7. Holding 4 c or 15 c
8. Volume 25 µl
9. Save Folder/file name
10. RUN
11. WAIT UNTIL THE LID TEMP reach 100oc.
12. Load PCR mix tube.
13. WAIT around 1.30 hour.
14. When it finish, click cancel.
15. Take PCR mix tube out.
16. WAIT UNTIL THE LID TEMP go down ̴ 30oc.
17. Turn off the PCR machine.

Storage:
2x Ampli Gold Taq master mix keep at -20oc or 4oc (often use).
PCR product transfer to 0.5 ml tube and keep at -20oc.
Electrophoresis:
1. Prepare 1.2% concentration gel.
2. Mix 1x TAE and agarose powder. 30 ml of 1xTAE with agarose powder (30 x 0.012)=0.36g
3. Measure agarose powder and put in flask then add 1x TAE until reach 30 ml.
4. Heat with microwave: time -> 30 sec then shake it and 30 sec again.
5. Add water until reach 30 ml.
6. Add 10,000x SYBR dye 3 µl (1 µl: 10 ml).
7. Pour mixer to plate and insert comb.
8. Wait until the gel is setted.
9. Mix PCR product (3 µl) and 10x buffer blue dye (1 µl). (Pipette dye to tube first then pipette
PCR product and then vortex and spin down)
10. Put gel in a tray and add 1x TAE until it cover the gel
11. Load marker first (2 µl) then load Mixer [PCR product (3 µl) and 10x buffer blue dye (1
µl)].
12. Set tray and machine: Close the cover and set 75 volt and run for 30-60 mins.

Gel image: Please wear gloves!

1. Turn on the computer and BIO-RAD machine (be sure the black cable is connected).
2. Put the gel on the tray;

3. Open image lab program.

 Agarose gel
 Position gel
 Move filter 1 (Move hand above machine)
 OK
 RUN protocol

4. Save or export as JPEG