Team:Caltech/Protocols

Biobrick Assembly Restriction Digest

For a double digest:
1. To a pcr tube, add 10 ul of miniprepped plasmid or purified PCR product
2. Also add 6 ul H2O.
3. Add 2 ul of the appropriate buffer (look at NEB's [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp Restriction Enzyme Activity chart])
4. Add 1ul of each enzyme.
5. Incubate at 37˚C for 1 hour.
6. Add CIP to the backbone digest. Incubate at 37˚C for 1 hour
7. PCR purify the reactions, unless you need to select out your part through gel extraction. Avoid these when possible, but if your insert is coming from a plasmid rather than a PCR, you need to select the insert you want and avoid having any of the leftover backbone in your ligation reaction. Also, you may need to do a triple digest to be able to size select for your insert. Use Geneious to find restriction sites.

Biobrick Assembly Ligation

1. In a PCR tube, add restriction digested and PCR purified insert to backbone in a 3-5:1 molar ratio, usually 1 ul backbone with 2 ul insert works.
2. Add 5.5 ul H20 (or other, so that the total volume of the ligation is 10 ul)
3. Add 1ul T4 ligase buffer
4. Add 0.5 ul T4 ligase
5. Create a negative control replacing the insert with water
6. Add reactions to thermal cycler. Incubate at 22˚C for 30 minutes, at 65˚C for 10 minutes to heat inactivate, and if not being used right away, leave at 4˚C.
7. Use 2 ul of the ligation reactions to transform cells.

Electrocompetent cells

1. Centrifuge 1 mL of the overnight E. coli culture to be transformed.
2. Pour off the supernatant in liquid waste container.
3. Add 1 mL of cold 10% glycerol.
4. Vortex the cells to resuspend them in the cold 10% glycerol.
5. Centrifuge the culture again and go to #2. Repeat 4-5 times. This step washes the cells with cold 10% glycerol to remove the LB.
6. Centrifuge the cells one final time, pour off the supernatant, add 50 microliters of cold 10% glycerol and vortex to resuspend.
7. Let the cells chill on ice for ~30 minutes.

Electroporation

1. Take 1 50ul aliquot of DH5a electrocompetent cells from -80˚C freezer.
2. Add 50 ul COLD 10% glycerol to dilute the cells, as ours are too concentrated. Divide into 2 50ul aliquots
3. Put electrocuvettes in freezer to cool. Keep on ice at all times before shocking
4. Add 1-2 ul of DNA (depends on source of DNA, use less for plasmids/biobricks, more for ligations etc.) to each 50 ul aliquot of cells.
5. Leave on ice for 2-5 minutes
6. Electroporate at 2.5 kV, 25 uF, 200 ohms by sliding cuvette into the machine and pressing the two buttons until you hear a beep
7. Immediately dilute with 250 ul LB or SOC. 8. Incubate in a shaker at 37˚C for 1 hour. 9. Plate using 50ul, otherwise you might get way too many colonies.

Enrichment cultures

• For BPA (since soluble)
  

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.

CopyControl Fosmid kit

http://www.epibio.com/pdftechlit/171pl1010.pdf
alternate link: http://arb-ls.com/products/copycontrol_fosmid_library_production_kit/171.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, or dilute 1:3 and use 1-3 ul of that to transform electrocompetent 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

X-Gal Plates

1 Dissolve X-Gal in DMSO at concentration of 20mg/ml (50x). Store at -20˚C. (There is a stock as of 8/24)
2 For typical 20 ml plate (with antibiotic already added in if needed), add 40 ul to the top of the plate and spread immediately with a L-shaped spreader to form an even coat.
3 Wait 30+ minutes until the X-Gal layer is dry.
4 Plate as usual. Colonies expressing beta-galactosidase (lacZ) will be blue.