| MegaBACE QC of ET Terminator Protocol |
| Version Number: | 2 |
| Start Production Date: | 11/01/99 |
| Author: | Jamie Jett |
| Edited by: | Susan Lucas |
| Reviewed by: | Paul Predki |
| Summary |
| Materials & Reagents |
| Materials/Reagents/Equipment | Vendor | Stock Number |
|---|---|---|
| Disposables | ||
| 96 well Cycle Plate | Robbins Scientific | 1055-90-0 |
| 50 mL Centrifuge Tube | Falcon | 2533-50 |
| 250 uL Pipet Tips | Rainin | RTL-250S |
| 1000uL Pipet Tips | Rainin | RT-2205 |
| aluminum foil | ||
| Stock Solutions | ||
| ET Terminator Kit (16 bottles) | Amersham | US81095 |
| pUC 19 DNA Standard(1000 ng/ul) | NEB | 304-1L |
| Primer - Forward -40M13 (250pmol/uL) GTT TTC CCA GTC ACG ACG TTG TA - Reverse -28M13 (250pmol/uL) AGG AAA CAG CTA TGA CCA T | GIBCO_Life Technologies | US81095 |
| Water (100%) | Millipore Milli-Q system | |
| Reagents | ||
| ET Terminator Kit - ET Terminator Premix - Formamide Loading Buffer | Amersham | US81095 |
| Ammonium Acetate (7.5M solution) | Sigma | A2706 |
| Equipment | ||
| MJ Thermocycler | MJ Research | |
| PE 9700 Thermocycler | Perkin Elmer | |
| P20 Pipet | Rainin | |
| P200 Pipet | Rainin | |
| P1000 Pipet | Rainin | |
| Eppendorf 5810 Centrifuge | Eppendorf | |
| 1L Graduated cylinder | VWR | |
| 200ml Graduated cylinder | VWR | |
| 2L Glass Bottle (2) | VWR | |
| Procedure |
Preparation of ET Sequencing Mixture
1. Cover 2-liter jars with aluminum foil. Label jars with ET Kit lot #, direction, Primer #, and date
2. Add 120 mL Milli-Q water and 3.2 mL of forward primer. Mix by swirling.
3. Add 640 mL ET Sequencing Kit. Mix well by swirling.
4. Aliquot 54 mL of forward sequencing mixture into 50 mL Falcon tubes. (Label tubes with ET Kit lot #, direction, Primer #)
5. Repeat steps 1-4 for reverse primer.
QC Plate of Sequencing Mix
A puc standard plates is used to determine the quality of Sequencing Mix
1. Add 5 uL puc standard to each well of 96 well plate
2. Add 5 uL of forward sequencing mixture in wells A, B ,C, D (1-12)
3. Add 5 uL of reverse sequencing mixture in wells E, F, G, H (1-12)
4. Centrifuge plate at 600 rpm for 15 seconds.
5. Place QC plate on thermocycler. Run standard ETMB cycle sequencing protocol.
6. Follow standard sequencing protocol.
QC Determination
1. Pass Criteria: 90% Pass Rate of a readlength over 450 bases
2. If below 90% Pass:
a) Determine if failure due to bad capillaries on Megabase sequencer. If apparent that a set of arrays is bad, sequencing mixtures pass QC.
b) If failure can not be narrowed to Megabase arrays:
1. Rerun same QC plate on another Megabase
OR
2. Prepare another QC plate of same sequencing mixtures. (Use same passing criteria)