Team:Hong Kong-CUHK/documentation.html
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<section id="protocol" class="group">
PROTOCOL<h1>
Contents |
Bacterial DNA extraction protocol for Azotobacter vinelandii or E.coli
Miniprep
Preparation of chemically competent BL21 E. coli cells
Day1
Streak Bl21 on a LB agar plate without antibiotic, grow overnight in 37℃ incubator
Day2
Pick a single colony and inoculate into 3ml LB broth, grow o/n in 37℃ shaker
Prepare & autoclave 500ml LB broth
Check if there is enough liquid nitrogen
Day3
Morning: pour the 3ml dense pre-culture into 500ml LB broth
Shake in 37C until OD600nm reach 0.8
[Melody's experience: it takes 4~5hrs]
Solution Needed
Wash Buffer I
800mM MgCl2 + 20mM CaCl2
Wash Buffer II
125mM CaCl2
Resuspend Buffer
85mM CaCl2 + 15% glycerol [filtered]
- pre-cool Wash Buffer I & Wash Buffer II in ice
- Pre-cool the centrifuge to 4C (with fixed angle rotor)
- Check the OD600nm of the 500ml culture
- Centrifuge the cells at 4000g for 5 mins, 4℃
- Discard the supernatant
- Gently resuspend the pellet in 20ml ice cold Wash Buffer I
- Put the samples on ice for 10 mins
- Centrifuge the cells at 4000g for 5 mins, 4℃
- Discard the supernatant
- Gently resuspend the pellet in 10ml ice cold Wash Buffer II
- Put the samples on ice for 10 mins
- Centrifuge the cells at 4000g for 5 mins, 4℃
- Discard the supernatant
- Resuspend cells in 20ml ice cold Resuspend Buffer
- Aliquot 200ul using sterile pre-chilled eppendorf tubes
Primer Design
Primers were designed manually using <a href="http://www.bioinformatics.org/sms2/pcr_primer_stats.html" target="_blank">PCR Primer Stats</a> of Sequence Manipulation Suite, for analyzing secondary structures and also annealing conditions.
PCR - Phusion DNA polymerase NEB
50ul reaction
Reactives | Volume(μL) |
---|---|
DNA template | 1 |
5x Phusion HF Buffer | 10 |
10mM dNTPs | 1.5 |
10uM Primer Fw | 0.5 |
10uM Primer Rv | 0.5 |
Phusion DNA polymerase | 0.25-0.5 |
100% DMSO | 1.5 |
dH2O | Up to 50 |
Total | 50 |
Cycling condition:
Initial Denaturation (1 cycle):
98°C 30 sec
Amplification (35 cycles):
98°C 10 sec
55-72 °C 30 sec
72°C 0.25- 0.5 min/kb
Final elongation (1 cycle)
72°C 3 min
</section>
<section id="notebook" class="group">
<h1>NOTEBOOKJUNE
- Preparation of Competent Cells of dh5α and BL21, aliquot the competent cells, stored in liquid nitrogen and then -80C refrigerator.
- Transformation of: J23100-119 into dh5α
- Preparation of special medium and plate for A. vinelandii
- Make LB agar solution
- Calibrate pH valve for N.P. and H.S. special medium
- Study the growth curve characteristics of A. vinelandii
- Study the natural antibiotics resistance of A. vinelandii
- Make plate with antibiotics -Ampicillin, kanamycin and chloramphenicol.
- Preparation of Competent Cells of A. vinelandii
- Purchase for primers for the Carbon fixation system project.
- Primer dilution of the all carbon fixation system primers
- Amplification by using PCR, following by run gel of A. vinelandii nifB gene.
- Amplification by using PCR, following by run gel of A. vinelandii nifQ gene.
- Amplification by using PCR, following by run gel of A. vinelandii FdxA gene.
- Amplification by using PCR, following by run gel of Aquifex aeolicus mbhS3 gene.
- Amplification by using PCR, following by run gel of Aquifex aeolicus mbhL3 gene.
- Amplification by using PCR, following by run gel of E. coli SH3PDZ domain gene.
JULY
- Make plate with 2% hard agar with antibiotics- chloramphenicol.
- Make plate with antibiotics -Ampicillin.
- Overlapping PCR of A. vinelandii nifB gene, A. vinelandii nifQ gene and A. vinelandii FdxA gene
- Restriction of Overlapping PCR product A. vinelandii nifB-nifQ-FdA, ligase into double terminator plasmid and transform into dh5α.
- Overlapping PCR of Aquifex aeolicus mbhS3 gene and mbhL3 gene.
- Restriction of Overlapping PCR product Aquifex aeolicus mbhS3-L3, ligase into double terminator plasmid and transform into dh5α.
- Make LB agar solution
- Restriction of PCR product E. coli SH3PDZ domain gene, ligase into C backbone and transform into dh5α.
- Stable genome integration of J23100 into A. vinelandii and study its characteristics
- Purchase for primers for the Nitrogen- repressible T7 expression system.
- Arrival of synthetic sequences of A. vinelandii nitrogenase structural gene
- Try Gibson assembly of synthetic sequences of A. vinelandii nitrogenase structural gene
AUGUST
- Arrival of synthetic sequences of A. vinelandii nitrogenase accessory gene.
- Arrival of synthetic sequences of Aquifex aeolicus hydrogenase accessory gene.
- Arrival of all primers for the Nitrogen- repressible T7 expression system
- Primer dilution of the all Nitrogen- repressible T7 expression system primers
- Amplification by using PCR, following by run gel of A. vinelandii nifH gene.
- Amplification by using PCR, following by run gel of A. vinelandii nifK gene
- Amplification by using PCR, following by run gel of A. vinelandii nifH gene with strong rbs.
- Amplification by using PCR, following by run gel of A. vinelandii nifH gene with weak rbs.
- Transformation of T7 RNA polymerase and T7 promoter into dh5α.
- Amplification by using PCR, following by run gel of BL21 T7 RNA polymerase.
- Overlapping PCR of A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase.
- Overlapping PCR of A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase.
- Amplification by using oligo PCR, following by run gel of random sequence
- Transformation of ptet- mRFP-d.t. and mRFP-d.t. into dh5α.
- Amplification by using oligo PCR, following by run gel of part of mRFP
- Restriction of PCR product random sequence, ligase into ptet- mRFP-d.t. plasmid and transform into dh5α.
- Transformation of amilcp-d.t. into dh5α.
- Restriction of PCR product nifH, ligase into mRFP-d.t. plasmid and transform into dh5α.
September
- Try Gibson Assembly of synthetic sequences of A. vinelandii nitrogenase accessory gene.
- Try Gibson Assembly of synthetic sequences of Aquifex aeolicus hydrogenase accessory gene.
- Restriction of PCR product A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase, ligase into double terminator plasmid and transform into dh5α.
- Restriction of PCR product A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase, ligase into double terminator plasmid and transform into dh5α.
- Restriction of biobrick A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase-double terminator ligase into random sequence with ptet-mRFP-d.t. and transform into dh5α.
- Restriction of biobrick A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase-double terminator ligase into random sequence with ptet-mRFP-d.t. and transform into dh5α.
- Restriction of PCR product A. vinelandii nifK gene,ligase into biobrick A. vinelandii nifH gene with strong rbs and BL21 T7 RNA polymerase-double terminator and random sequence with ptet-mRFP-d.t. and transform into dh5α.
- Restriction of PCR product A. vinelandii nifK gene,ligase into biobrick A. vinelandii nifH gene with weak rbs and BL21 T7 RNA polymerase-double terminator and random sequence with ptet-mRFP-d.t. and transform into dh5α.
- Restriction of PCR product A. vinelandii nifK gene, ligase into C back bone and transform into dh5α.
- Restriction of PCR product A. vinelandii nifH gene, ligase into C back bone and transform into dh5α.
- Restriction of PCR product part of mRFP, ligase into C back bone and transform into dh5α.
- Restriction of BBa_K1314013, ligase into A backbone and transform into dh5α.
- Restriction of BBa_K1314014, ligase into A backbone and transform into dh5α.
- Restriction of PCR product part of mRFP, ligase into amilcp- d.t. and transform into dh5α.
- Restriction of PCR product, random sequence, ligase into T7 promoter and transform into dh5 α.
- Restriction of biobrick random sequence- T7 promoter, ligase into C backbone and transform into dh5α
- Restriction of biobrick random sequence- T7 promoter, ligase into amilcp- d.t.-part of mRFP, and transform into dh5α.
- Send all biobricks to sequencing
- Transformation of BBa_K1314011 into BL21.
- Stable genome integration of BBa_K1314013 into A. vinelandii
- Stable genome integration of BBa_K1314014 into A. vinelandii
- Stable genome integration of BBa_K1314011 into A. vinelandii
- Blunt end ligation and transformation to make BBa_K1314015
October
</section>
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