Biomod/2013/Harvard/protocols: Difference between revisions
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===Agilent Herculase II PCR Amplification=== | ===Agilent Herculase II PCR Amplification=== | ||
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=== Bacterial Cell Culture Solutions === | === Bacterial Cell Culture Solutions === | ||
====LB media==== | |||
# For 1L of LB media add: | |||
## 10g Tryptone | |||
## 10g NaCl | |||
## 5g Yeast extract | |||
## Fill to 1L with DI water | |||
# Autoclave at P6 setting | |||
====LB-Agar Plate media==== | |||
# For 1L of LB-Agar media, mix: | |||
## 960mL water (860 mL if adding glucose) | |||
## 10g Tryptone | |||
## 10g NaCl | |||
## 5g Yeast extract | |||
## 15g Agar (add agar last) | |||
# Autoclave (liquid option): 30 min. sterilization / 20 min drying | |||
# Cool to 50˚C in water bath | |||
# Next three steps are under flame | |||
# (if adding glucose: add 100mL of 20% glucose, final conc. 2% glucose) | |||
# Add antibiotics to the specified final conc: | |||
## chloramphenicol: 50 µg/mL | |||
## ampicillin: 100 µg/mL | |||
## Tetracycline: 50 µg/mL | |||
# Pour onto the petri dishes (pre-marked by type: two-black lines=CM, red-line=glucose) | |||
===BLACaM Assays === | ===BLACaM Assays === |
Revision as of 06:57, 18 June 2013
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Protocols
Agilent Herculase II PCR Amplification
Protocol:
- In a PCR tube, mix (50 µL final volume):
- 35 µL water
- 10 µL HercII Buffer (5X)
- 0.5 µL dNTP (25mM each, 100mM total final)
- 1.25 µL forward primers (final conc?)
- 1.25 µL reverse primers (final conc?)
- 1 µL Template DNA (diluted to 10 ng/µL)
- 1 µL HercII DNA polymerase
- Give the tubes a brief spin
- Thermocycling conditions:
- Step 1: 95˚C, 2:00
- Step 2 X30
- Phase 1: 95˚C, 0:15
- Phase 2: (Tm - 5)˚C, 0:20
- Phase 3: 72˚C, 0:30 per 1kb
- Step 3: 72˚C, 3:00
ZYMO DNA Clean & Concentrator
ZYMO DNA Clean & Concentrator - 5 Kit was used to purify DNA from PCR.
Protocol:
- Add 2-7 Volumes of DNA Binding Buffer to each volume of DNA sample.
- For plasmid or genomic DNA 2kb, add 2 volumes.
- For PCR or short DNA fragments, add 5 volumes.
- For ssDNA purification, add 7 volumes
- Load the mixture into a Zymo-Spin Column in a Collection Tube.
- Centrifuge at full speed (≥10,000 x g) for 30 seconds. Discard flow through.
- Add 200 µl of DNA Wash Buffer to the column and centrifuge for 30 seconds.
- Repeat previous step, this time centrifuging for 1 minute.
- Place the Zymo-Spin Column into a new 1.5 ml tube. Add ≥6 µl of DNA Elution Buffer or water directly to the column matrix and spin (30 seconds) to elute the DNA.
Bacterial Cell Culture Solutions
LB media
- For 1L of LB media add:
- 10g Tryptone
- 10g NaCl
- 5g Yeast extract
- Fill to 1L with DI water
- Autoclave at P6 setting
LB-Agar Plate media
- For 1L of LB-Agar media, mix:
- 960mL water (860 mL if adding glucose)
- 10g Tryptone
- 10g NaCl
- 5g Yeast extract
- 15g Agar (add agar last)
- Autoclave (liquid option): 30 min. sterilization / 20 min drying
- Cool to 50˚C in water bath
- Next three steps are under flame
- (if adding glucose: add 100mL of 20% glucose, final conc. 2% glucose)
- Add antibiotics to the specified final conc:
- chloramphenicol: 50 µg/mL
- ampicillin: 100 µg/mL
- Tetracycline: 50 µg/mL
- Pour onto the petri dishes (pre-marked by type: two-black lines=CM, red-line=glucose)
BLACaM Assays
Chemically Competent Cells Transformation
This protocol uses NEB Chemically Competent Cells (ie Turbo Competent E. coli High Efficiency)
Protocol:
- Thaw chemically competent cells on ice.
- Transfer 50 µl of competent cells to a 1.5 ml microcentrifuge tube (if necessary).
- Add 2 µl of assembled product to NEB competent cells.
- Mix gently by pipetting up and down or flicking the tube 4-5 times. Do not vortex. Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at 42°C for 30 seconds. Do not mix.
- Transfer tubes on ice for 2 minutes.
- Add 950 µl of room temperature SOC media to tubes.
- Place the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
- Warm selection plates to 37°C.
- Spread 100 µl of the cells onto the plates with appropriate antibiotics. Use amp plates for positive control sample.
- Incubate plates overnight at 37°C.