Recommended Cycle Sequencing Protocols For ABI 3100
Template Quantity
Template |
Quantity |
PCR product:
- 100-200bp
- 200-500bp
- 500-1000bp
- 1000-2000bp
- >2000bp
|
- 1-3 ng
- 3-10 ng
- 5-20 ng
- 10-40 ng
- 20-50 ng
|
Single-stranded |
- 25-50 ng
|
Double-stranded |
- 150-300 ng
|
Cosmid, BAC |
- 0.5-1.0 mg
|
Bacterial genomic DNA |
- 2-3 mg
|
| Reaction Mixtures |
|
|
Reagent |
Full Reaction |
Half Reaction |
Half Reaction(10 μl) |
Big Dye Premix |
8 μl |
4 μl |
2 μl |
Big Dye Seq. Buffer |
- |
2 μl |
1 μl |
Template |
See Table above |
See Table above |
See Table above |
Primer (10 μM) |
1 μl |
1 μl |
1 μl |
Water |
q.s. |
q.s. |
q.s. |
Total |
20 μl |
20 μl |
10 μl |
Primer Quantity
Primer needed = 3.2 - 10 pmoles
PCR Cycle Sequencing Settings for Big Dye V3.1
Initial denaturing |
96°C |
1min |
25 cycles of |
96°C |
10sec |
50°C |
5sec |
60°C |
4min |
Hold at |
4°C |
|
Ethanol/EDTA Precipitation to clean up reactions
- Add 5uL of 125 mM EDTA. Make sure the EDTA reaches the bottom of the tube.
- Add 60uL of 100% ethanol to each tube.
- Finger vortex and incubate at room temperature for 15 min.
- Spin samples in a microcentrifuge at max speed in 4 ° C for 20min.
- Carefully aspirate off the supernatant.
- Add 60 μl of 70% Ethanol.
- Spin samples in a microcentrifuge at maximum speed at 4 ° C for 15 minutes.
- Aspirate off the supernatant.
- Dry sample for 15 minutes in a Speed-Vac (longer if air-drying). Protect samples from light while they are drying.
This Web page is maintained by Julie B. Wolf, UMBC;
Last updated on
3/2/2010
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