Team:Caltech/Protocols
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[[Team:Caltech/Notebook|Back to Timeline]] . [[Team:Caltech/Recipes|Recipes for Mixes]]<br/><br/> | [[Team:Caltech/Notebook|Back to Timeline]] . [[Team:Caltech/Recipes|Recipes for Mixes]]<br/><br/> | ||
+ | ===Biobrick Assembly Restriction Digest=== | ||
+ | For a double digest:<br/> | ||
+ | 1. To a pcr tube, add 10 ul of miniprepped plasmid or purified PCR product<br/> | ||
+ | 2. Also add 6 ul H2O.<br/> | ||
+ | 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])<br/> | ||
+ | 4. Add 1ul of each enzyme. <br/> | ||
+ | 5. Incubate at 37˚C for 1 hour.<br/> | ||
+ | 6. Add CIP to the backbone digest. Incubate at 37˚C for 1 hour<br/> | ||
+ | 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.<br/> | ||
- | + | ===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.<br/> | |
+ | 2. Add 5.5 ul H20 (or other, so that the total volume of the ligation is 10 ul)<br/> | ||
+ | 3. Add 1ul T4 ligase buffer<br/> | ||
+ | 4. Add 0.5 ul T4 ligase<br/> | ||
+ | 5. Create a negative control replacing the insert with water<br/> | ||
+ | 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.<br/> | ||
+ | 7. Use 2 ul of the ligation reactions to transform cells.<br/> | ||
- | < | + | ===Electrocompetent cells=== |
- | + | 1. Centrifuge 1 mL of the overnight E. coli culture to be transformed.<br/> | |
+ | 2. Pour off the supernatant in liquid waste container.<br/> | ||
+ | 3. Add 1 mL of cold 10% glycerol.<br/> | ||
+ | 4. Vortex the cells to resuspend them in the cold 10% glycerol.<br/> | ||
+ | 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.<br/> | ||
+ | 6. Centrifuge the cells one final time, pour off the supernatant, add 50 microliters of cold 10% glycerol and vortex to resuspend.<br/> | ||
+ | 7. Let the cells chill on ice for ~30 minutes.<br/> | ||
- | < | + | ===Electroporation=== |
+ | 1. Take 1 50ul aliquot of DH5a electrocompetent cells from -80˚C freezer.<br/> | ||
+ | 2. Add 50 ul COLD 10% glycerol to dilute the cells, as ours are too concentrated. Divide into 2 50ul aliquots<br/> | ||
+ | 3. Put electrocuvettes in freezer to cool. Keep on ice at all times before shocking<br/> | ||
+ | 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.<br/> | ||
+ | 5. Leave on ice for 2-5 minutes<br/> | ||
+ | 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<br/> | ||
+ | 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.<br/> | ||
+ | |||
+ | ===Enrichment cultures=== | ||
* For BPA (since soluble)<br/> | * For BPA (since soluble)<br/> | ||
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.<br/> | 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.<br/> | ||
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1. Set up two flasks: one with vitamin media, one without vitamin.<br/> | 1. Set up two flasks: one with vitamin media, one without vitamin.<br/> | ||
2. Add small amounts (around 50mL or 50mg) of the ten LA river samples into each flask.<br/> | 2. Add small amounts (around 50mL or 50mg) of the ten LA river samples into each flask.<br/> | ||
- | For both: culture initially for 3 days, then reculture for 7 days. Then test for DNA and continue cultures.<br/ | + | For both: culture initially for 3 days, then reculture for 7 days. Then test for DNA and continue cultures.<br/> |
- | + | ===CopyControl Fosmid kit=== | |
- | http://www.epibio.com/pdftechlit/171pl1010.pdf</ | + | http://www.epibio.com/pdftechlit/171pl1010.pdf<br/> |
+ | alternate link: http://arb-ls.com/products/copycontrol_fosmid_library_production_kit/171.pdf<br/> | ||
- | + | ===Gibson Assembly (Adapted from Cambridge 2010)=== | |
0a. PCR DNA strands (50uL rxn)<br/> | 0a. PCR DNA strands (50uL rxn)<br/> | ||
0b. DpnI digest and purify products (elute w/ 20-30uL EB or H<sub>2</sub>O);(Nanodrop)-> normally 50-120ng/nL<br/> | 0b. DpnI digest and purify products (elute w/ 20-30uL EB or H<sub>2</sub>O);(Nanodrop)-> normally 50-120ng/nL<br/> | ||
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2. To 3uL of DNA, add 7.5uL of Gibson Mix<br/> | 2. To 3uL of DNA, add 7.5uL of Gibson Mix<br/> | ||
3. Incubate @ 50C for 30-60 minutes with heated lid<br/> | 3. Incubate @ 50C for 30-60 minutes with heated lid<br/> | ||
- | 4. Cool, then transform into chemically competent cells | + | 4. Cool, then transform into chemically competent cells, or dilute 1:3 and use 1-3 ul of that to transform electrocompetent cells.<br/> |
- | + | ===Mobio PowerMax Soil kit=== | |
- | http://www.mobio.com/images/custom/file/protocol/12988-10.pdf | + | http://www.mobio.com/images/custom/file/protocol/12988-10.pdf<br/> |
- | + | ===p450 binding assay, organic extraction for analysis by HPLC=== | |
- | + | 1. Obtain a ~80mM solution of the chemicals in DMSO, 1mL total.<br/> | |
- | + | 2. Form a 200uL solution of the diluted substrate, p450, NADP<sup>+</sup>, glucose, glucose dehydrogenase, and buffer. (see [[Team:Caltech/Recipes|Mixes]])<br/> | |
- | + | 3. Leave overnight for reaction.<br/> | |
- | + | 4. Add 200uL water and 180uL DCM; vortex thoroughly.<br/> | |
- | + | 5. Centrifuge briefly and pipette out the organic (bottom) layer into a new tube.<br/> | |
- | + | 6. Repeat steps 3 and 4.<br/> | |
- | + | 7. Evaporate liquid from the new tube on 40°C plate.<br/> | |
- | + | 8. Add 50uL of .5x diluted ACN, then centrifuge at 12,000 RPM for 5 min.<br/> | |
- | + | 9. Place the 50uL samples into HPLC tube and bottle.<br/> | |
+ | 10. Run HPLC for 10 min per sample.<br/> | ||
- | < | + | ===p450 binding assay, organic extraction for analysis by GCMS=== |
+ | 1. Obtain a ~80mM solution of the chemicals in DMSO, 1mL total.<br/> | ||
+ | 2. Form a 200uL solution of the diluted substrate, p450, NADP<sup>+</sup>, glucose, glucose dehydrogenase, and buffer. (see [[Team:Caltech/Recipes|Mixes]])<br/> | ||
+ | 3. Leave 4 hours for reaction.<br/> | ||
+ | 4. Add 300uL buffer and 180uL DCM; vortex thoroughly.<br/> | ||
+ | 5. Centrifuge briefly and pipette out the organic (bottom) layer into a new tube.<br/> | ||
+ | 6. Repeat steps 3 and 4, but with 500uL DCM (to dilute sample for GCMS).<br/> | ||
+ | 7. Run GCMS.<br/> | ||
+ | |||
+ | ===Pulse Gel Field Electrophoresis=== | ||
PFGE separation of 0.5 µg of Lambda Mono Cut Mix, 0.1% agarose gel, 0.5X TBE<br/> | PFGE separation of 0.5 µg of Lambda Mono Cut Mix, 0.1% agarose gel, 0.5X TBE<br/> | ||
Parameters: 6 V/cm, 15°C for 20 hours.<br/> | Parameters: 6 V/cm, 15°C for 20 hours.<br/> | ||
- | Switch times ramped from 0.5-1.5 seconds. | + | Switch times ramped from 0.5-1.5 seconds.<br/> |
- | + | ===Phusion PCR=== | |
Thermocycling conditions:<br/> | Thermocycling conditions:<br/> | ||
Initial Denaturation: 98°C for 30 seconds <br/> | Initial Denaturation: 98°C for 30 seconds <br/> | ||
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* 55°C, 60°C, 65°C for 15 seconds | * 55°C, 60°C, 65°C for 15 seconds | ||
* 72°C for 15 seconds | * 72°C for 15 seconds | ||
- | Final Extension: 72°C for 5 minutes | + | Final Extension: 72°C for 5 minutes<br/> |
- | + | ===Qiagen Miniprep kit=== | |
- | www.qiagen.com/hb/qiaprepminiprep | + | www.qiagen.com/hb/qiaprepminiprep<br/> |
- | + | ===Transforming DNA from Distribution Plates:=== | |
1. Thaw competent cells on ice.<br/> | 1. Thaw competent cells on ice.<br/> | ||
2. Add 10 microliters of pure water to each well of DNA from plates, pipette up and down.<br/> | 2. Add 10 microliters of pure water to each well of DNA from plates, pipette up and down.<br/> | ||
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7. Heat Shock for 45 sec by using a water bath set to 42°C and then chill on ice for 2 min.<br/> | 7. Heat Shock for 45 sec by using a water bath set to 42°C and then chill on ice for 2 min.<br/> | ||
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.<br/> | 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.<br/> | ||
- | 9. For source plate DNA, plate 100 microliters. | + | 9. For source plate DNA, plate 100 microliters.<br/> |
- | + | ===Taq PCR (16s insert)=== | |
Initial denaturation: 94°C for 1:30 min<br/> | Initial denaturation: 94°C for 1:30 min<br/> | ||
35 cycles of:<br/> | 35 cycles of:<br/> | ||
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*54°C for 1:00 min | *54°C for 1:00 min | ||
*72°C for 2:00 min | *72°C for 2:00 min | ||
- | Final extension: 72°C for 6:00 min</ | + | Final extension: 72°C for 6:00 min<br/> |
+ | |||
+ | ===Colony PCR (for ~.7kb insert)=== | ||
+ | Suspend colonies in 10 ul H20<br/> | ||
+ | Lyse with 98°C incubation for 10 minutes<br/> | ||
+ | Use 1 ul of this suspension as template<br/> | ||
+ | Set up tubes with 7 ul H20, 1 ul template, 1 ul forward primer, 1 ul reverse primer, 10 ul Phusion master mix <br/> | ||
+ | Cycle: <br/> | ||
+ | 2 minutes at 98°C<br/> | ||
+ | Run 30 cycles of:<br/> | ||
+ | *10 seconds at 98°C | ||
+ | *30 seconds at 55°C-65°C | ||
+ | *40 seconds at 72°C | ||
+ | Final extension: 5 minutes at 72°C<br/> | ||
+ | |||
+ | ===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)<br/> | ||
+ | 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 an L-shaped spreader to form an even coat.<br/> | ||
+ | 3 Wait 30+ minutes until the X-Gal layer is dry.<br/> | ||
+ | 4 Plate as usual. Colonies expressing beta-galactosidase (lacZ) will be blue.<br/> | ||
}} | }} |
Latest revision as of 23:00, 28 September 2011
Project |
Back to Timeline . Recipes for Mixes Biobrick Assembly Restriction DigestFor a double digest: Biobrick Assembly Ligation1. 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. Electrocompetent cells1. Centrifuge 1 mL of the overnight E. coli culture to be transformed. Electroporation1. Take 1 50ul aliquot of DH5a electrocompetent cells from -80˚C freezer. 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.
1. Set up two flasks: one with vitamin media, one without vitamin. CopyControl Fosmid kithttp://www.epibio.com/pdftechlit/171pl1010.pdf Gibson Assembly (Adapted from Cambridge 2010)0a. PCR DNA strands (50uL rxn) Mobio PowerMax Soil kithttp://www.mobio.com/images/custom/file/protocol/12988-10.pdf p450 binding assay, organic extraction for analysis by HPLC1. Obtain a ~80mM solution of the chemicals in DMSO, 1mL total. p450 binding assay, organic extraction for analysis by GCMS1. Obtain a ~80mM solution of the chemicals in DMSO, 1mL total. Pulse Gel Field ElectrophoresisPFGE separation of 0.5 µg of Lambda Mono Cut Mix, 0.1% agarose gel, 0.5X TBE Phusion PCRThermocycling conditions:
Final Extension: 72°C for 5 minutes Qiagen Miniprep kitwww.qiagen.com/hb/qiaprepminiprep Transforming DNA from Distribution Plates:1. Thaw competent cells on ice. Taq PCR (16s insert)Initial denaturation: 94°C for 1:30 min
Final extension: 72°C for 6:00 min Colony PCR (for ~.7kb insert)Suspend colonies in 10 ul H20
Final extension: 5 minutes at 72°C X-Gal Plates1 Dissolve X-Gal in DMSO at concentration of 20mg/ml (50x). Store at -20˚C. (There is a stock as of 8/24)
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