Silver: Ligation: Difference between revisions
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'''Using NEB T4 DNA ligase''': | |||
#Mix: | |||
#*50-100 ng de-phosphorylated, [[Silver: Restriction_Digest|digested]] vector DNA | |||
#*3-10x molar excess [[Silver: Restriction_Digest|digested]] insert DNA | |||
#*2 μL 10x T4 DNA ligase buffer | |||
#*x μL distilled water (final volume of reaction should be 20 μL) | |||
#*1 μL T4 DNA ligase | |||
#Mix thoroughly, incubate for 30 minutes at room temperature | |||
#Proceed to [[Silver: Bacterial_Transformation|transformation]]. Store excess ligation mixture at -20 °C | |||
'''Using Roche's [http://www.roche-applied-science.com/fst/amplification.htm?/sis/amplification/pifs/cloning/rapid_ligation_kit.htm Rapid DNA Ligation Kit], stored at -20 °C.''' <br> | |||
Keep the total mass of DNA below 200 ng. | Keep the total mass of DNA below 200 ng. | ||
#Mix: | #Mix: | ||
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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 (2x T4 DNA ligase buffer) or 2uL of 10x 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. |
Latest revision as of 09:09, 2 July 2010
Using NEB T4 DNA ligase:
- Mix:
- Mix thoroughly, incubate for 30 minutes at room temperature
- Proceed to transformation. Store excess ligation mixture at -20 °C
Using 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 (2x T4 DNA ligase buffer) or 2uL of 10x 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.