PCR Amplification for Sequencing
| Version Number: | 1a |
| Development Date: | 8-15-99 |
| Start RND date: | 9-01-99 |
| Author: | Chris Detter, Damian Scott |
| Reviewed by: | Paul Richardson |
Summary
To amplify clones directly from glycerol stocks using PCR and Exo-SAP as a clean-up method prior to sequencing.
Materials & Reagents
| Materials/Reagents/Equipment | Vendor | Stock Number |
|---|---|---|
| Disposables | ||
| MJ V2.0 384 well PCR plate | MJ Research | MSP-3852 |
| Stock Solutions | ||
| 95% ETOH | AAPER | N/A |
| 1.4% Agarose solution | JGI | N/A |
| Reagents | ||
| PCR nucleotide | Amersham | US77212 |
| Taq Polymerase | Amersham | 27-0799-63 |
| dNTP set, 100mM soln's | Amersham | 27-2035-03 |
| Exonuclease 1 2500 Units | USB | 70073 Z |
| Phosphatase, alkaline, shrimp | Roche | 1 758 250 |
| Equipment | ||
| IKA-Vibrax-VXR plate shaker | IKA | N/A |
| 384 well 1ul Pin Tool | VSP | N/A |
| MJ PTC-225 Tetrad Thermocycler | MJ Research | N/A |
| Sonicator | VRW | 150-HT |
| Eppendorf 5810 Centrifuge | Eppendorf | 5810 |
Procedure
1)Pull glycerol plates and defrost at room temperature
2)Once defrosted, place glycerol plates on IKA-Vibrax-VXR plate shaker (medium setting for at least 2 minutes)
3)Prepare PCR reaction mix
4)On ice aliquot 10ul/well of PCR cocktail into 384 MJ V2.0 PCR plate.
5)Use Pin Tool to transfer DNA from glycerol plates into 384 MJ V2.0 PCR plate on ice
-Sterilize Pin Tool by placing in Sonicator for 20 seconds
-Dip Pin Tool into 95% ETOH and flame it
-Cool Pin Tool on benchtop for 1 minute
-Take glycerol plate off plate shaker
-Dip pin tool into glycerol plate and then dip into destination plate
-Perform the previous step twice for each plate
-Repeat all steps for each plate
6)Centrifuge MJ V2.0 PCR plate up to 1000rpm and back down
7)Thermocycle conditions:
Hot start (place PCR plate on thermocycler once temperature reaches 94C)!!!
a)94 C 4 min. Hold
b)94 C 30 sec.
c)55 C 30 sec.
d)68 C 4 min. (+10 sec./cycle)
e)4 C Forever Hold
35 cycles of steps b to d
7)Once cycle is completed, prepare Exo-SAP reaction on ice
8)Add 3ul/well of Exo-SAP to the PCR reaction plate on ice
9)Centrifuge MJ V2.0 PCR plate up to 1000rpm and back down
10)Place spun down plate in MJ thermocycler under the following conditions for Exo-SAP:
37 C Hold 45 minutes
80 C Hold 10 minutes
4 C Hold forever
11)Quantitate using a 1.4%Agarose gel with ETBR
-use bromophenol blue with 50% glycerol in water for loading dye
-add 5ul of exo-sap product and 5ul loading dye to each well
-use 1KB ladders and 125ng, 250ng, and 500nglambda standards
-run at 120V for 20 minutes
12)Seqeunce using ET Terminator Amersham chemistry
Reagent/Stock Preparation
PCR cocktail(prepare in a Falcon 50 ml tube on ice)
| For 1 reaction | For 12-384 well plates | |
| 8.5 ul Milli-Q H20 | 44800 ul Milli-Q H20 | |
| 1.0 ul 10X PCR buffer | 5280 ul 10X PCR buffer | |
| 0.2 ul 10mM dNTP’s | 1056 ul 10mM dNTP’s | |
| 0.14ul pUC Fwd 10uM primer | 739.2ul pUC Fwd 10uM primer | |
| 0.14ul pUC Rv 10uM primer | 739.2ul pUC Rv 10uM primer | |
| 0.07ul Amersham Taq | 369.6ul Amersham Taq | |
| Total 10ul/well | Total 10ul/well |
Exo-SAP preparation (prepare in a 15ml Falcon tube on ice)
| For 1 reaction | For 12-384 well plates | |
| Milli-Q water 2.49ul | Milli-Q water 13147.2 ul | |
| 1M Tris pH8.0 0.03ul | 1M Tris pH8.0 158.4 ul | |
| Exonuclease 1 0.18ul | Exonuclease 1 950.4 ul | |
| SAP 0.3ul | SAP 1584ul | |
| Total 3ul/well | Total 3ul/well |
Questions or Comments: Detter2@llnl.gov
