Julius B. Lucks/Protocols: Difference between revisions
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== Pre-Cut Oligo Phosphorylation == | |||
Ingredients: | |||
* 1 uL primer | |||
* 1 uL 10x ligase buffer (black striped aliquat in freezer) | |||
* 7.5 uL water | |||
* 0.5 uL PNK (enzyme freezer - modifying enzyme T4 PNK) | |||
# Combine ingredients (one tube for each primer) in a tube and incubate at 37 C for 1 hr. | |||
# Combine the 2 tubes, and add 180 uL water | |||
# Take this tube and boil for 5 min on a hot plate in a beaker of water (boil water in a beaker and put the tube in the boiling water) | |||
# Take beaker off hot plate and put on bench to ramp down to room temperature | |||
Use this solution as you would for an insert in a ligation. | |||
== Glucose Minimal Media == | == Glucose Minimal Media == | ||
* Ammonium Sulfate (NH4)2SO4 - 0.502g (0.0038 mol) | * Ammonium Sulfate (NH4)2SO4 - 0.502g (0.0038 mol) | ||
Revision as of 15:46, 27 June 2008
Pre-Cut Oligo Phosphorylation
Ingredients:
- 1 uL primer
- 1 uL 10x ligase buffer (black striped aliquat in freezer)
- 7.5 uL water
- 0.5 uL PNK (enzyme freezer - modifying enzyme T4 PNK)
- Combine ingredients (one tube for each primer) in a tube and incubate at 37 C for 1 hr.
- Combine the 2 tubes, and add 180 uL water
- Take this tube and boil for 5 min on a hot plate in a beaker of water (boil water in a beaker and put the tube in the boiling water)
- Take beaker off hot plate and put on bench to ramp down to room temperature
Use this solution as you would for an insert in a ligation.
Glucose Minimal Media
- Ammonium Sulfate (NH4)2SO4 - 0.502g (0.0038 mol)
- MW 132.14 g/mol
- CAS 7783-20-2
- Dibasic Potassium Phosphate KH2PO4*3H2O - 2.244g (0.01 mol)
- MW 228.23
- CAS
- Monobasic Potassium Phosphate K2HPO4 - 5.226g (0.038 mol)
- MW 136.09
- CAS 7778-77-0
- Sodium Citrate - Na3C6H5O7 - 0.25g (0.00085 mol)
- HOC(COONa)(CH2COONa)2*2H2O
- MW 294.10
- CAS
- Dextrose Anhydrous (glucose) - 1.0g (0.0056 mol)
- CH2OH(CHOH)4CHO
- MW 180.16
- CAS 50-99-7
- Thiamine (0.1% stock) - 0.25ml (xxx mol)
- MW
- CAS
- Magnesium Sulfate MgSO4*7H2O (1M stock) - 0.5ml (0.0005 mol)
- MW 246.48
- CAS 10034-99-8
- H2O - up to 0.5L
Glycerol Minimal Media
Same as Glucose Minimal Media except substitute for Dextrose Anhydrous
- Glycerol C3H8O3 (50% stock) - 1.04 ml (0.52 g = 0.0056 mol)
- MW 92.09
- CAS 56-81-5
