V. Preparation of Sequencing Template for Plasmids.

A. TempliPhi DNA Amplification (This protocol is from Amersham Biosciences TempliPhi DNA Sequencing Template Amplification Kit (25-6400-01)

1. Reaction mix File name:

Chemicals	Per reaction	Total volume	8 reactions	Check
Denature buffer (410480)	5µl		40µl
DNA template	2µl
TempliPhi premix (410402)	5µl		40µl
Bacteriophage phi 29 DNA polymerase (410478)	0.2µl		1.6µl
Total	12.2µl

2. Protocol

1. Thaw the TempliPhi premix and denature buffer on ice. Vortex gently to ensure mixing.
2. Dispense 5µl aliquots of denature buffer into an appropriate reaction tube.
3. Transfer samples to the denature buffer. (1ul of glycerine stock or 1 toothpick colony)
4. Heat the tubes at 95ºC for 3 min and then cool on ice.
5. Add 5µl of TempliPhi premix and 0.2µl polymerase to the cooled sample. Vortex briefly to mix.
6. Incubate at 30ºC for 16 hours in GeneAmp PCR system 9700 (Program F5-RCA). Start: ______finish: _______.
7. Heat-inactivate the enzyme by incubating at 65ºC for 10 min. Cool to 4ºC.
8. Store the amplified DNA at -20ºC or 4ºC.

3. Restriction Digestion of RCA products

Chemical	Standard reaction	8 reactions	Check
Rnase/Dnase free water	2.5µl	22.5
BSA (10X) Promega R396E: 1mg/ml	1µl	9
Reaction buffer H (10X) Promega R008A	1µl	9
RCA product	5µl
EcoR I (12unit/µl) Promega R601A 2.5-3 unit/µg of DNA to be cut	0.5µl	4.5
Total	10µl

a. Add 10µl water to RCA product.

b. Mix the reactions by pipetting before and after enzyme is added.

c. Incubate the reactions in water bath at 37°C for 1hour.

d. Store at -20°C

4. Sample record sheet

No.	Sample #	Remark
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20
Conclusion: