WORKSHOPS PROTOCOLS

ANALYSIS METHOD OF GENETIC POLYMORPHISM

POLYMERASE CHAIN REACTION (PCR)

FETUIN-A (AHSG) C766G GENE POLYMORPHISM

1. Sample
: DNA
2. Primer
: Fetuin A- forward and reverse
3. Reaction mixture :
- PCR master mix
- Fetuin forward primer

15L
1L

- Fetuin reverse primer

- dH2O
- DNA
4. PCR Condition

1L
13L
2L

Green PCR Master Mix contain:

1. Taq Polymerase
2. PCR buffer

3. dNTPs
4. MgCl2
5. Loading buffer

94 7 min
94 1 min
58 1 min
72 1 min

35 cycles

72 7 min
4 ---

ELECTROPHORESIS FOR FETUIN PCR PRODUCT

1. Place 2% of agarose gel into electrophoresis tank with appropriate
buffer. Buffer used is 1 X Tris-Acetic-EDTA (TAE).
2.
3.
4.
5.
6.

Pipet into the well 5L of PCR products, marker and non template control.
Turn on electricity. Set voltage: 100 V.
Set timer: 30 min. Let the samples move until line number 5.
Turn off electrophoresis.
Take the gel from electrophoresis tank.

(Use glove since gel and buffer contain ethidium bromide)

7. Check the result under UV transluminator (GelDoc).
8. Make a photograph.
9. Fetuin (AHSG = alpha(2) Heremans-Schmid glycoprotein) PCR product is showed

by a band of 405 bp.

RESTRICTION FRAGMENT LENGTH POLYMORPHISM

Samples: PCR Product of Fetuin gene
1. Add into microtube:
- PCR product

5L

- 10X buffer
1L
- SacI
0.5L
- MQ
3.5L
2. Mix gently with pipetting.
3. Incubate at 37 for overnight/16 hours.
4. Electrophoresis of RFLPs products on 3% of agarose gel.
5. Allele C does not contain SACI site (405 bp), wheres allele G yields 193 and 212 bp
fragments.