Back to Timeline . Recipes for Mixes
Biobrick Assembly Ligation
http://www.neb.com/nebecomm/products/protocol446.asp
Biobrick Assembly Restriction Digest
http://www.neb.com/nebecomm/products/protocol445.asp
Enrichment cultures
1. Set up 16 tubes: 8 tubes with vitamin media vs. 8 tubes with media (no vitamin), 4 tubes for each of the four locations.
2. Place 8 test tubes in 30°C shaker and 8 test tubes in room temperature shaker.
- For 17α-estradiol, DDT, and nonylphenol (since non-soluble)
1. Set up two flasks: one with vitamin media, one without vitamin.
2. Add small amounts (around 50mL or 50mg) of the ten LA river samples into each flask.
For both: culture initially for 3 days, then reculture for 7 days. Then test for DNA and continue cultures.
Fosmid kit
http://www.epibio.com/pdftechlit/171pl1010.pdf
Gibson Assembly (Adapted from Cambridge 2010)
0a. PCR DNA strands (50uL rxn)
0b. DpnI digest and purify products (elute w/ 20-30uL EB or H2O);(Nanodrop)-> normally 50-120ng/nL
1. Mix DNA
2. To 3uL of DNA, add 7.5uL of Gibson Mix
3. Incubate @ 50C for 30-60 minutes with heated lid
4. Cool, then transform into chemically competent cells
Mobio PowerMax Soil kit
http://www.mobio.com/images/custom/file/protocol/12988-10.pdf
p450 binding assay, organic extraction for analysis by HPLC
1. Obtain a ~80mM solution of the chemicals in DMSO, 1mL total.
2. Form a 200uL solution of the diluted substrate, p450, NADP+, glucose, glucose dehydrogenase, and buffer. (see Mixes)
3. Leave overnight for reaction.
4. Add 200uL water and 180uL DCM; vortex thoroughly.
5. Centrifuge briefly and pipette out the organic (bottom) layer into a new tube.
6. Repeat steps 3 and 4.
7. Evaporate liquid from the new tube on 40°C plate.
8. Add 50uL of .5x diluted ACN, then centrifuge at 12,000 RPM for 5 min.
9. Place the 50uL samples into HPLC tube and bottle.
10. Run HPLC for 10 min per sample.
p450 binding assay, organic extraction for analysis by GCMS
1. Obtain a ~80mM solution of the chemicals in DMSO, 1mL total.
2. Form a 200uL solution of the diluted substrate, p450, NADP+, glucose, glucose dehydrogenase, and buffer. (see Mixes)
3. Leave 4 hours for reaction.
4. Add 300uL buffer and 180uL DCM; vortex thoroughly.
5. Centrifuge briefly and pipette out the organic (bottom) layer into a new tube.
6. Repeat steps 3 and 4, but with 500uL DCM (to dilute sample for GCMS).
7. Run GCMS.
Pulse Gel Field Electrophoresis:
PFGE separation of 0.5 µg of Lambda Mono Cut Mix, 0.1% agarose gel, 0.5X TBE
Parameters: 6 V/cm, 15°C for 20 hours.
Switch times ramped from 0.5-1.5 seconds.
Phusion PCR
Thermocycling conditions:
Initial Denaturation: 98°C for 30 seconds
25-35 cycles:
- 98°C for 10 seconds
- 55°C, 60°C, 65°C for 15 seconds
- 72°C for 15 seconds
Final Extension: 72°C for 5 minutes
Qiagen Miniprep kit
www.qiagen.com/hb/qiaprepminiprep
Transforming DNA from Distribution Plates:
1. Thaw competent cells on ice.
2. Add 10 microliters of pure water to each well of DNA from plates, pipette up and down.
3. Transfer into storage tube.
4. Pipette 1-2 microliters of the DNA into the competent cell tubes.
5. Stir with pipette tip, gently flick tube.
6. Leave on ice for 30 minutes.
7. Heat Shock for 45 sec by using a water bath set to 42°C and then chill on ice for 2 min.
8. Pipette 500 micro Liters of S.O.C. (LB + glucose) into 14ml culture tubes; transfer the competent cells into these tubes and incubate in a 37 degree shaker for 0-60 minutes before plating.
9. For source plate DNA, plate 100 microliters.
Taq PCR (16s insert)
Initial denaturation: 94°C for 1:30 min
35 cycles of:
- 94°C for 0:30 min
- 54°C for 1:00 min
- 72°C for 2:00 min
Final extension: 72°C for 6:00 min
Colony PCR (for ~.7kb insert)
Suspend colonies in 10 ul H20
Lyse with 98°C incubation for 10 minutes
Use 1 ul of this suspension as template
Set up tubes with 7 ul H20, 1 ul template, 1 ul forward primer,1 ul reverse primer, 10 ul Phusion master mix
Cycle:
2 minutes at 98°C
Run 30 cycles of:
- 10 seconds at 98°C
- 30 seconds at 55°C-65°C
- 40 seconds at 72°C
Final extension: 5 minutes at 72°C
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