7,287
edits
Line 22: | Line 22: | ||
Vortex | Vortex | ||
== | ==Procedure== | ||
===10μ | ===10μL Ligation Mix=== | ||
''Larger ligation mixes are also commonly used'' | ''Larger ligation mixes are also commonly used'' | ||
*1.0 μL 10X T4 ligase buffer | *1.0 μL 10X T4 ligase buffer | ||
*6:1 | *6:1 molar ratio of insert to vector (~10ng vector) | ||
*Add (8.5 - vector and insert volume)μl ddH<sub>2</sub>O | *Add (8.5 - vector and insert volume)μl ddH<sub>2</sub>O | ||
*0.5 μL T4 Ligase | *0.5 μL T4 Ligase | ||
Line 32: | Line 32: | ||
<math> {Insert\ Mass\ in\ ng} = 6\times\left[\frac{{Insert\ Length\ in\ bp}}{{Vector\ Length\ in\ bp}}\right]\times{Vector\ Mass\ in\ ng} </math> | <math> {Insert\ Mass\ in\ ng} = 6\times\left[\frac{{Insert\ Length\ in\ bp}}{{Vector\ Length\ in\ bp}}\right]\times{Vector\ Mass\ in\ ng} </math> | ||
''' | '''The insert to vector molar ratio can have a significant effect on the outcome of a ligation and subsequent transformation step. Molar ratios can vary from a 1:1 insert to vector molar ratio to 10:1. It may be necessary to try several ratios in parallel for best results.''' | ||
===Method=== | ===Method=== | ||
#Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube | #Add appropriate amount of deionized H<sub>2</sub>O to sterile 0.6 mL tube | ||
Line 44: | Line 45: | ||
#Use disks shiny side up | #Use disks shiny side up | ||
#Store at -20°C | #Store at -20°C | ||
==Critical steps== | ==Critical steps== |
edits