Silver: Ligation: Difference between revisions
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#*4-10x molar excess [[Silver: Restriction_Digest|digested]] insert DNA. | #*4-10x molar excess [[Silver: Restriction_Digest|digested]] insert DNA. | ||
#*2 µL tube #2 (DNA dilution buffer) | #*2 µL tube #2 (DNA dilution buffer) | ||
#*distilled water to final volume of 10 | #*distilled water to final volume of 10 µL total volume | ||
#Add 10 µL of freshly mixed tube #1 (T4 DNA ligase buffer). | #Add 10 µL of freshly mixed tube #1 (T4 DNA ligase buffer). | ||
#Add 1 µL tube #3 (T4 DNA ligase). | #Add 1 µL tube #3 (T4 DNA ligase). | ||
#Mix well, incubate for 15 min. at room temperature. | #Mix well, incubate for 15 min. at room temperature. | ||
#Proceed to [[Silver: Bacterial_Transformation|transformation]]. Store excess ligation mixture at -20 °C. | #Proceed to [[Silver: Bacterial_Transformation|transformation]]. Store excess ligation mixture at -20 °C. |
Revision as of 08:31, 1 August 2006
Use Roche's Rapid DNA Ligation Kit, stored at -20 °C.
Keep the total mass of DNA below 200 ng.
- Mix:
- Add 10 µL of freshly mixed tube #1 (T4 DNA ligase buffer).
- Add 1 µL tube #3 (T4 DNA ligase).
- Mix well, incubate for 15 min. at room temperature.
- Proceed to transformation. Store excess ligation mixture at -20 °C.