Silver:Restriction digest protocol
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Solutions/reagents:
- <a name="restriction enzyme1">restriction enzyme1
<tab>(20 units/µl)</a> - <a name="restriction enzyme2">restriction enzyme2
<tab>(20 units/µl)</a> - 10X restriction buffer
- 10X BSA
- PCR product DNA
- distilled H2O
- <a name="BioBrick vector">BioBrick vector
<tab>(BBa_V0002 or BBa_V0100)</a> - <a name="CIP">CIP
<tab>(10 units/µl)</a>
Parameters:
- V - Volume of PCR product DNA obtained (µl).
- C - Concentration of BioBrick vector (µg/µl).
Equipment:
- Incubator
- Reaction tubes
Steps:
- Digestion of PCR product
Use the following table as a checklist for preparing the reaction in reaction tube (1):
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Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.PCR product DNA 10X BSA 10X restriction buffer restriction enzyme1 restriction enzyme2 distilled H2O Restriction Digest - PCR product V 3.5 µl 3.5 µl 0.2 µl 0.2 µl Make up the volume to 35 µl - Incubate at 37°C for at least 1 hr.
Incubation overnight is better. - Purify digested insert using PCR product purification kit.
- Digestion of BioBrick vector: Recommended procedure
Use the following table as a checklist for preparing the reaction in reaction tube (2):
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Note: keep the glycerol concentration below 5%, the volume of both restriction enzymes added should not exceed 5% of the total reaction volume.BioBrick vector 10X BSA 10X restriction buffer restriction enzyme1 restriction enzyme2 distilled H2O Restriction Digest - vector 7/C 1 µl 1 µl 0.2 µl 0.2 µl Make up the volume to 10 µl - Incubate at 37°C for 12 hrs(overnight).
- Measure out 0.1 µl of <a href="#CIP" >CIP</a> into reaction tube (2).
Incubate at 37°C for 1 hr.
TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 14 hrs, 0 mins
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