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Team:OUC-China/Lab note
From 2013.igem.org
Lab-note
Week1
June 26th
Person: Wenjun Wang
Experiment:
1. Get the AMB-1 bacteria strain from Nanjing University.
2. Keep the strain in 30℃.
Week2
June 30th
Person: Wengjun Wang
Experiment:
1. PCR: Get 16srDNA sequence from AMB-1 bacteria genome to detect the strain.
2. Agarose electrophoresis: detect the PCR product.
3. Transform: transform the ligastion product into Top 10 to prepare for sequence.
Week3
July 13th ~ 15th
Person: Wenjun Wang
Experiment:
1. Get the AMB-1 bacteria strain from Nanjing University.
2. Keep the strain in 30℃.
Week4
June 30th
Person: Kaili Qin
Experiment:
1. Equip the medium: Equip the medium of Magnetospirillum Magneticum AMB-1.
July 20th
Person: Wenjun Wang
Experiment:
1. PCR: Get mamJ sequence from PCR product to finish the Over-Lap PCR.
2. Agarose electrophoresis: detect the plasmid PCR product.
3. Transform: transform the ligastion product into Top 10 to prepare for sequence.
4. PCR: Get mamK sequence from PCR product to finish the Over-Lap PCR.
5. Agarose electrophoresis: detect the PCR product.
6. Product purification: purify the PCR product.
7. PCR: Get mamAB sequence from AMB-1 bacteria genome.Person: Qianyun Lu
1. Culture the E.coli cells: Psb1c3.
Week5
July 21st
Person: Wenjun Wang
Experiment:
1. Agarose electrophoresis: detect the plasmid PCR product.
2. PCR: Get mamB sequence from PCR product.
3. PCR: Get mamQ sequence from PCR product.
4. PCR: Get mamJ sequence from PCR product to finish the Over-Lap PCR.
5. Agarose electrophoresis: detect the plasmid PCR product.
6. Product purification: purify the PCR product.Person: Yu Wang
Experiment:
1. Bacterination: E.coli with part K1059003-B0035-GFPlva-K1059004-B0015 pSB1AK3 as backbone; E.coli with RFP, pSB1C3 as backbone.Person:Kaili Qin
Experiment:
1. Plasmid miniprep: plasmid Extraction of PSB1C3 for 5 tubes.
2. Electrophoresis: gel is good.
3. Equip the medium: Equip the medium of LB (solid).Person:Qianyun Lu
Experiment:
1. PCR production purification: 16SrRNA from AMB-1.
2. SDS page gel electrophoresis: the purified PCR production.Person:Xiaodong Zhong
Experiment:
1. PCR genome for mamI gene & PCR purification.
2. PCR genome for mamB gene.July 22nd
Person:Xue Sun
Experiment:
1. Plasmid miniprep: Plasmid pSB1AK3 with K1059003-B0035-GFPlva-K1059004-B0015, and pSB1C3 with RFP.
2. Enzyme digestion.
3. Gel extraction.
4. Recombination: Get part K1059003-B0035-GFPlva-K1059004-B0015, pSb1C3 as backbone.Person:Kaili Qin
Experiment:
1.Enzyme digestion: enzyme digestion of PSB1C3 for 25ul system.
2.Gel recovery: gel recovery of the product of enzyme digestion.
3.Bacterination: bacterinate PSB1C3-RFP, RNA1 into tubes of 10ml.
4.Pick bacteria: pick bacteria of PSB1C3-RFP for conservation.
5.Genomic DNA extraction: extracting genomic DNA of E.coli.
6. Electrophoresis: gel is bad.
7. Bacterination: bacterinate PSB1C3-RFP, RNA1 into tubes of 10ml for 4 tubes.Person:Qianyun Lu
Experiment:
1. Ligation: ligate the purified PCR production with T vector.
2. Transformation of Top10.July 23rd
Person:Qianyun Lu
Experiment:
1. Extraction of genomic DNA from E.coli.
2. Pick the bacteria and culture the E.coli cells for four hours.
3. PCR: the cultured E.coli cells.
4. SDS page gel electrophoresis: PCR production.Person:Kaili Qin
Experiment:
1. Plasmid miniprep: plasmid Extraction of PSB1C3 -RFP for 5 tubes.
2. Enzyme digestion: enzyme digestion of PSB1C3 for 50ul system of 3 tubes, use EcoR1, Pst1.3. Electrophoresis: gel is good.
4. Genomic DNA extraction: extracting genomic DNA of E.coli.
5. Electrophoresis: gel is bad.
6. Gel recovery: gel recovery of PSB1C3.
7. Electrophoresis: gel is good for 50ng/ul each.
8. Bacterination: bacterination of E.coli for 6 tubes.Person:Qiu Wang
Experiment:
1. Transformation: Plate cultivation for 14h.July 24th
Person:Qianyun Lu
Experiment:
1. Extraction of genomic DNA from E.coli.
2. PCR: the cultured E.coli cells.Person:Kaili Qin
Experiment:
1. Genomic DNA extraction: extracting genomic DNA of E.coli.
2. Electrophoresis: gel is bad.Person:Wenjun Wang
Experiment:
1. Agarose electrophoresis: detect the plasmid PCR product.
2. PCR: Get mamI sequence from PCR product.
3. PCR: Get mamL sequence from PCR product.
4. PCR: Get mamK sequence from PCR product to finish the Over-Lap PCR.
5. Agarose electrophoresis: detect the plasmid PCR product.
6. Product purification: purify the PCR product.Person:Yu Wang
Experiment:
1. Pick bacteria and PCR examining, the aim fragment is 1409bp.
2. Sequencing.July 25th
Person:Wenjun Wang
Experiment:
1. PCR: Try to get mamAB gene cluster sequence from AMB-1 bacterial strain.
2. Get the genome of AMB-1 with TaKaRa MINI Best bacteria genome DNA Extraction Ver2.0.
3. PCR: Try to get mamAB gene cluster sequence from AMB-1 genome.
4. Agarose electrophoresis: detect the plasmid PCR product.Person:Xue Sun
Experiment:
1. d1 to d6 as primer PCR, get part K1059005.
2. Purification.
3. Recombine PCR product to pMD-19 T vector.
4. Transformation.Person:Kaili Qin
Experiment:
1.Equip the medium：Equip the medium of AMB-1.Person:Qianyun Lu
Experiment:
1. SDS page gel electrophoresis: PCR production and the genomic DNA.Person:Xiaodong Zhong
Experiment:
1. Transformation of E.coli (top10):
Part: mamK+T vector
Part: mamI+T vector
Part: mamL+T vector
Part: mamQ+T vector
Part: mamB+T vector (failure)July 26th
Person:Yu Wang
Experiment:
1. Blue-white selection.
2. PCR examining, the aim fragment is 388bp.
3. Sequencing.Person:Kaili Qin
Experiment:
1. Bacterination: bacterinate PSB1C3-RFP into tubes of 10ml.July 27th
Person:Qiu Wang
Experiment:
1. Measuring Growth curve, E.coli with RFP, pSB1C3 as plasmid backbone.Person:Qianyun Lu
Experiment:
1. Extraction of genomic DNA from E.coli.
2. SDS page gel electrophoresis: the genomic DNA.
Week6
July 28th
Person:Qiu Wang
Experiment:
1. Measure Growth curve.Person:Kaili Qin
Experiment:
1. Bacterination: bacterinate PSB1C3-RFP, E.coil with single-1 protection sequence into tubes of 10ml.Person:Qianyun Lu
Experiment:
1. Culture the E.coil cells: PSB1C3.July 29th
Person:Yu Wang
Experiment:
1. Bacterial culturing.Person:Wenjun Wang
Experiment:
1. PCR: Get mamB sequence from AMB-1 bacterial strain.
2. PCR: Get mamJ sequence from AMB-1 bacterial strain.
3. PCR: Get mamE sequence from AMB-1 bacterial strain.
4. PCR: Get mamE sequence from AMB-1 bacterial strain.
5. Agarose electrophoresis: detect the DNA digestion product.Person:Kaili Qin
Experiment:
1. Plasmid miniprep: plasmid Extraction of PSB1C3-RFP, E.coil with single-1 protection sequence for 2 tubes each.
2. Enzyme digestion: enzyme digestion of PSB1C3 and E.coil with single-1 protection sequence for 25ul system of 2 tubes each, use EcoR1, Pst1.
3. Electrophoresis: gel is good.Person:Qianyun Lu
Experiment:
1. Miniprep: Psb1c3.
2. Digestion: digest the plasmid with EcoRI and Pst-HF.
3. SDS page gel electrophoresis.
4. Gel extraction of DNA.July 30th
Person:Qiu Wang
Experiment:
1. Lstwenmin1 to Lstwenmin4 as primer PCR, get part K1059006.
2. Purification.
3. Recombine PCR product to pMD-19 T vector.
4. Transformation.Person:Kaili Qin
Experiment:
1. Transform: transform the ligastion (single-1 protection sequence-PSB1C3) product into Top 10.
2. Bacterial coating.July 31st
Person:Xue Sun
Experiment:
1. Blue-white selection.
2. PCR and electrophoresis examining, the aim fragment is 388bp and 210.
3. Sequencing.
4. The PCR result a fragment at 120bp, so we failed the part k1059006. We designed another two primers and be ready for more PCR.Person:Kaili Qin
Experiment:
1. Pick bacteria: pick bacteria for PCR.
2. PCR: use PCR to detect the bacteria we want (single-1 protection sequence-PSB1C3)), 25ul system.Person:Qianyun Lu
Experiment:
1. Glycerol stocks: Psb1c3, 2 tubes.
2. Miniprep: Psb1c3.
3. Digestion: digest the plasmid with EcoRI and Pst-HF.
4. Gel extraction of DNA.August 1st
Person:Yu Wang
Experiment:
1. Bacterination.
2. E.coil with part K1059005 PMD-19 T vector as backbone.Person:Xiaodong Zhong
Experiment:
1. PCR genome for mamB, mamJ, mamE, mamC gene.Person:Wenjun Wang
Experiment:
1. PCR: Get mamC sequence from AMB-1 bacterial strain.
2. Agarose electrophoresis: detect the mamC PCR product.
3. Product purification: purify the PCR product.
4. PCR: Get the mamC sequence which is more nicety from the product of product purification.August 2nd
Person:Xue Sun
Experiment:
1. Plasmid minprep.
2. Enzyme digestion.
3. Recombination: Get part K1059005-B0035-GFPlva-K1059004-B0015, pSB1C3 as backbone.Person:Xiaodong Zhong
Experiment:
1. Transformation of E.coil (top10): mamB+T vector.Person:Wenjun Wang
Experiment:
1. PCR：Get mamB, mamE sequence from AMB-1 bacterial strain.
2. Get the genome of AMB-1.
3. Transform: transform the ligastion product into Top 10.August 3rd
Person:Qiu Wang
Experiment:
1. Transformation and Bacterination.Person:Qianyun Lu
Experiment:
1. Miniprep: Psb1c3, 6 tubes.
2. Digestion: digest the plasmid with EcoRI and Pst-HF, XbaI and Pst-HF.
3. SDS page gel electrophoresis.
4. Gel extraction of DNA.Person:Xiaodong Zhong
Experiment:
1. E. coli Cell Culture: Top10 (mamB+T vector) Resistance: Ampicillin Medium: LB.
2. mamK PCR purification: Get mamK gene.
3. PCR genome for mamB: 16x50ul PCR system. Get mamB gene.Person:Wenjun Wang
Experiment:
1. Agarose electrophoresis: detect the PCR product.
2. Get the genome of AMB-1.
3. Agarose electrophoresis: detect the genome product.
4. PCR: to detect the product of transform.
5. PCR: Get mamC sequence from AMB-1 bacterial strain.
6. PCR: Get 16srDNA sequence from AMB-1 bacterial strain to detect the strain.
7. Agarose electrophoresis: detect the genome product.
