Standard Operating Procedure

 

Real-time PCR with SYBR-Green

 

Hazards:

 

 

Protection:

Wear gloves

 

Waste:

 

Spill Clean-Up:

 

Procedure:

 

Set-up Reactions

Notes:

• Use Qiagen QuantiTect SYBR Green PCR kit (contains SYBRGreen, Hotstart Taq polymerase, dNTP mix, PCR buffer, and MgCl₂).
• Set-up reactions in special PCR set-up area on the bench next to Evil Tetramer. This area is for PCR set-up only, do not bring amplified product back to this area. Spray down bench with RNase Away.
• Keep reagents on ice.

• Need to make stock mixture fresh before I’m ready to run each set of samples.
• Keep samples cold, use cold block from 6-274.
• Initially mix together in 1.5 ml eppendorf tube, then place reaction in special real-time PCR tubes.
• Spin down tubes in special mini-centrifuge by the cycler.

 

Master mix (25 µl)	Set-up Tube #1 (x 1 reactions)	Final set-up in Real-Time PCR Tube (24 µl stock mix + 1 µl template)
2X QuantiTect SYBR Green PCR Master Mix	12.5 µl	-------
F primer (20uM)	0.4 µl	-------
R primer (20uM)	0.4 µl	-------
RNase-free water	10.7 µl	-------
cDNA	-------	1 µl

 

Program Protocol

HPRT (Hotstart)

• Stage 1: Hold at 95ºC for 900 s.
• Stage2-3-Temperature Cycle, repeat for 40 times
• 94ºC for 15 s
• 58ºC for 30 s
• 72ºC for 30 sec with OPTICS ON
• Stage 3: Melt Curve 55ºC to 95ºC at 0.2/sec with Optic Ch1 ON

 

Forward Primer:

Reverse Primer:

Probe: SYBRGreen

Expected fragment size: (HPRT 101 bp)

Controls: no template

 

Smart Cycler

1. Create run (060204 John Doe).
2. Change dye set to appropriate dye (SYBRGreen).
3. Add/remove sites depending on number of reactions (up to 32 reactions).
4. Determine protocol for each site (can be the same or different between sites).
5. Change sample ID to appropriate name (OT-I DP HPRT).
6. Chose graphs (SYBR threshold, 2^nd Derivative, Melt).
7. Run PCR.

 

Analysis (after PCR has completed)

1. Under analysis settings, change the threshold value (look at 2^nd Derivative graph, move threshold line as close as possible to 2^nd derivative lines).
2. Change from primary curve to 2^nd Derivative curve
3. Update analysis settings.

 

 

Troublshooting

• Always test primers using traditional PCR (if more than one band-cannot use for real-time PCR).
• Determine optimal annealing temperature. Run a real-time PCR, changing temperature from 58ºC to 60ºC to 62ºC and altering extension times (30, 45, 50, 60s)
• When analyzing data, look at the 2^nd derivative peak. The peak should be sharp rather than rounded. Also look at the primary curve, a dramatic inflection is best.
• Reproducibility of the data is important-should be able to repeat 3X with the same results.

 

Alternative set-up without using the Qiagen Kit

 

Master mix (26 ml)	Set-up Tube #1 (x1 reactions)	Set-up Tube #2 (x1 reactions)	Final set-up in Real-Time PCR Tube (24 µl stock mix + 1 µl template)
ddH2O	13.15	-------	-------
F primer (20uM)	-------	0.4	-------
R primer (20uM)	-------	0.4	-------
Probe (SG) (2uM stock)	1.25	-------	-------
MgCl2 (25mM)	2	-------	-------
10X PCR buffer	2.6	-------	-------
dNTPs (1.25mM)	4	-------	-------
Taq	0.2	-------	-------
cDNA	-------	-------	1