"
Team:OUC-China/Lab note
From 2013.igem.org
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.
Week7
August 4th
Person:Kaili Qin
Experiment:
1. Ligation: J23101 order vector with chloramphenicol resistance; double2 order
RBS-GFPLVA- single2-terminator and vector with chloramphenicol resistance.
2. Transform: transform the ligastion product into Top 10.
3. Bacterial coating
Person:Yu Wang
Experiment:
1. PCR: Get BBa_K1059006 sequence from PCR product.
2. Agarose electrophoresis: detect the PCR product.
3. Product purification: purify the PCR product.
4. Ligation: recombine PCR product to pMD-19 T vector.
5. Sequencing
6. Transformation: transform the ligastion product into Top 10.
Person:Qianyun Lu
Experiment:
1. SDS page gel electrophoresis.
August 5th
Person:Qiu Wang
Experiment:
1. Blue -white selection.
2. PCR and electrophoresis examining.
3. PCR and electrophoresis examining, get the aim fragment.
4. Sequencing.
Person:Qianyun Lu
Experiment:
1. Culture the E.coli cells: pSB1C3.
Person:Kaili Qin
Experiment:
1. Pick bacteria: pick bacteria for PCR.
2. PCR: use pcr to detect the bacteria we want.(double2-RBS-GFPLVA-single2-terminator-PSB1C3), 25ul system.
3. Electrophoresis.
4. Send sequencing.
5. Conservation: conservate E.coli with.double2-RBS-GFPLVA-single2-terminator-PSB1C3 plasmid.
August 6th
Person:Xue Sun
Experiment:
1. Plasmid minprep.
Person:Qianyun Lu
Experiment:
1. Miniprep: pSB1C3.
2. Digestion: digest the plasmid with XbaI and Pst-HF.
3. SDS page gel electrophoresis.
4. Gel extraction of DNA.
5. PCR production purification: J23106+RBS, mamQ, mamL, B0015.
6. SDS page gel electrophoresis: J23106+RBS, mamQ, mamL, B0015.
100bp, J23106+RBS, mamQ, mamL, B0015
7. PCR amplification: J23106+RBS, B0015.
8. SDS page gel electrophoresis: J23106+RBS, B0015.
Person:Wenjun Wang
Experiment:
1. PCR: Get mamJ sequence from PCR product and add prefix and suffix onto the sequence.
2. PCR: Get mamQ sequence from PCR product and add prefix and suffix onto the sequence.
3. Agarose electrophoresis: detect the PCR product.
4. Product purification: purify the PCR product.
5. Ligation: BBa_J23101-protection order 1-RBS-GFPLVA-terminator and vector with chloramphenicol resistance.
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 Xba1, Pst1.
3. Electrophoresis: gel is good.
August 7th
Person:Xue Sun
Experiment:
1. Bacterination.
Person:Wenjun Wang
Experiment:
1. Transform: transform the ligastion product into Top 10.
2. PCR: Get mamL sequence from PCR product and add prefix and suffix onto the sequence.
3. PCR: Get mamQ sequence from PCR product and add prefix and suffix onto the sequence.
4. Agarose electrophoresis: detect the PCR product.
5. PCR: Get J23106+B0032 sequence from PCR product and add prefix and suffix onto the sequence.
6. PCR: Get B0015 sequence from PCR product and add prefix and suffix onto the sequence.
7. Agarose electrophoresis: detect the PCR product.
8. Product purification: purify the PCR product.
Person:Kaili Qin
Experiment:
1. Gel recovery: gel recovery of pSB1C3.
2. Electrophoresis: gel is good for 30ng/ul each.
3. PCR: use PCR to B0015 cloning, 50ul system.
4. Electrophoresis: gel is good.
5. PCR Purification: PCR Purification of B0015.
6. Electrophoresis: gel is good for 30ng/ul each.
7. Enzyme digestion: enzyme digestion of RBS-mamL for 25ul system, use Xba1, Pst1;RBS-mamL for 25ul system, use Spe1; B0015 for 25ul system, use Xba1; promoter-RBS for 25ul system, use EcoR1, Pst1.
8. Ligation: RBS-mamL order pSB1C3; promoter-RBS order pSB1C3; RBS-mamL order B0015.
9. Transform: transform the ligastion product into Top 10.
10. Bacterial coating.
11. Bacterination: bacterinate E.coli with PSB1C3-RFP into tubes of 10ml.
Person:Qianyun Lu
Experiment:
1. PCR amplification: mamL, three tubes.
2. PCR production purification: mamL, three tubes; J23106+RBS, three tubes.
3. SDS page gel electrophoresis: mamL, three tubes; J23106+RBS, three tubes.
J23106+RBS, mamL
4. Digestion: digest RBS+ mamL with XbaI and Pst-HF, digest RBS+mamL with SpeI, digest B0015 with XbaI, digest J23106+RBS with EcoRI and Pst-HF.
5. SDS page gel electrophoresis.
6. Ligation: ligate RBS+mamL with Psb1c3, ligate J23106+RBS with pSB1C3,ligate RBS+ mamL with B0015.
August 8th
Person:Qiu Wang
Experiment:
1. Miniprep the pSB1C3, J23101, RGL-K1059005.
August 9th
Person:Yu Wang
Experiment:
1. Enzyme digestion: BBa_J23101 with EcoR I and SpeI, BBa_ K1059002 with XbaI and PstI, BBa_K1059005 with EcoR I and PstI, pSB1C3 with EcoRI and PstI.
2. Gel extraction pSB1C3 as vector.
Person:Qianyun Lu
Experiment:
1. Miniprep: pSB1C3, ten tubes.
2. Digestion: three tubes are digested with XbaI and Pst-HF, two tubes are digested with EcoRI and Pst-HF.
3. SDS page gel electrophoresis.
4. Gel extraction of DNA.
5. PCR amplification: mamQ, four tubes.
6. SDS page gel electrophoresis.
7. PCR production purification: mamQ, two tubes.
8. SDS page gel electrophoresis.
9. Digestion: digest J23106+RBS with EcoRI and SpeI, digest RBS+mamQ with XbaI and Pst-HF.
10. PCR amplification: RBS+mamL, four tubes.
Person:Kaili Qin
Experiment:
1. Pick bacteria: pick bacteria for PCR.
2. PCR: use pcr to detect the bacteria we want(promoter-RBS-PSB1C3, RBS-mamL-PSB1C3), 25ul system.
3. Enzyme digestion: enzyme digestion of PSB1C3 for 50ul system, 3 tubes, use Xba1, Pst1; pSB1C3 for 25ul system of 2 tubes, use EcoR1, Pst1.
4. PCR: use pcr to cloning RBS-mamL-B0015, 50ul system.
5.Electrophoresis: gel is good.
6. Gel recovery: gel recovery of pSB1C3.
7. Electrophoresis: gel is good for 20ng/ul each.
Electrophoresis of PSB1C3 vector
August 10th
Person:Xue Sun
Experiment:
1. Recombination.
Person:Qianyun Lu
Experiment:
1. SDS page gel electrophoresis.
2. PCR amplification: J23106+RBS, two tubes; B0015, two tubes.
3. PCR production purification: RBS+mamL, four tubes.
4. SDS page gel electrophoresis.
5. Digestion: digest RBS+mamL with SpeI.
6. Culture the E.coli cells: JB11.
7. PCR production purification: mamI.
8. SDS page gel electrophoresis.
Person:Wenjun Wang
Experiment:
1. Product purification: purify the PCR product, J1.
2. Plastid extraction: B0015, J2310, C+.
3. Digestion: use enzyme XbaI, Pst I to get part B0015, a double terminator.
4. Ligation: mamK onto pMB-19 T, prepare for sequence.
5. PCR: amplification mamJ sequence from PCR product.
6. Agarose electrophoresis: detect the PCR product.
7. Get part for agrose, mamJ.
8. PCR: Get mamI sequence from PCR product and add prefix and suffix onto the sequence.
9. Agarose electrophoresis: detect the PCR product.
10. Product purification: purify the PCR product.
Person:Kaili Qin
Experiment:
1. PCR Purification: PCR Purification of mamL1~4.
2. Conservation: promoter-RBS-PSB1C3 (P4), RBS-mamL-PSB1C3 (L4).
3. Ligation: RBS-mamJ, RBS-mamI, RBS-mamB order PMD-19 vector separately.
Week8
August 11th
Person:Yu Wang
Experiment:
1. Transformation.
2. Plate cultivation for 14h.
Person:Kaili Qin
Experiment:
1. PCR: use PCR to detect mamQ1~10, 25ul system.
2. Electrophoresis: gel is bad.
3. Transform: transform the ligastion product ( RBS-mamJ-T vector;RBS-mamI-T vector, RBS-mamB-T vector, RBS-mamK-T vector) into Top 10.
Person:Qianyun Lu
Experiment:
1. Miniprep: JB11, B0015, each two tubes.
2. Digestion: digest JB11 with EcoRI and SpeI, digest B0015 with XbaI and Pst-HF, digest RBS+mamL with XbaI and SpeI.
3. Ligation: ligate RBS+J23106 with pSB1C3, ligate RBS+mamL and BOO15 with pSB1C3.
August 12th
Person:Xue Sun
Experiment:
1. Pick bacteria and PCR examining
Person:Kaili Qin
Experiment:
1. Transform: transform the ligastion product(RBS-mamL-B0015-PSB1C3; promoter-RBS-PSB1C3) into Top 10.
2. Bacterial coating.
3. Pick bacteria: pick bacteria we want(RBS-mamL-B0015-PSB1C3, promoter-RBS-mamQ-PSB1C3).
Person:Qianyun Lu
Experiment:
1. Pick the bacteria and culture the E.coli cells for four hours.
2. PCR: the cultured E.coli cells.
August 13th
Person:Qianyun Lu
Experiment:
1. PCR: the cultured E.coli cells.
2. SDS page gel electrophoresis.
3. Ligation: ligate the purified mamB and mamL with T vector.
4. PCR amplification: mamB, mamK, mamJ.
5. SDS page gel electrophoresis.
Person:Qiu Wang
Experiment:
1. Sequencing.
August 14th
Person:Qianyun Lu
Experiment:
1. PCR amplification: mamJ.
2. SDS page gel electrophoresis.
Person:Kaili Qin
Experiment:
1. Pick bacteria: pick bacteria we want(RBS-mamL-B0015-PSB1C3, promoter-RBS-mamQ-PSB1C3).
Person:Yu Wang
Experiment:
1. Bacterination.
2. Make competent cells.
August 15th
Person:Wenjun Wang
Experiment:
1. PCR: get mamJ sequence from AMB-1 bacterial strain.
2. Agarose electrophoresis: detect the plasmid PCR product.
3. Product purification: purify the PCR product.
4. PCR: get mamC sequence from AMB-1 bacterial strain.
Person:Qiu Wang
Experiment:
1. Miniprep.
2. Make gel.
Person:Qianyun Lu
Experiment:
1. SDS page gel electrophoresis.
2. PCR amplification: mamL.
3. PCR production purification: RBS+mamL.
4. SDS page gel electrophoresis.
Person:Kaili Qin
Experiment:
1. Ligastion: mamJ, mamK order PMD19-T vector.
August 16th
Person:Xue Sun
Experiment:
1. Enzyme digestion.
Person:Kaili Qin
Experiment:
1. PCR: use PCR to detect mamK-pMD19-T vector1~15, 25ul system.
Person:Qianyun Lu
Experiment:
1. PCR amplification: mamL.
2. SDS page gel electrophoresis.
August 17th
Person:Wenjun Wang
Experiment:
1. PCR: amplification mamC sequence from PCR product.
2. Agarose electrophoresis: detect the plasmid PCR product.
3. Product purification: purify the PCR product.
4. PCR: amplification mamI sequence from PCR product.
5. PCR: amplification mamJ sequence from PCR product.
6. Agarose electrophoresis: detect the plasmid PCR product.
7. Product purification: purify the PCR product.
8. PCR: amplification mamB sequence from PCR product.
9. Agarose electrophoresis: detect the plasmid PCR product.
10. Product purification: purify the PCR product.
Person:Qianyun Lu
Experiment:
1. SDS page gel electrophoresis.
2. PCR amplification: mamQ, two tubes.
3. PCR production purification: RBS+mamL.
4. SDS page gel electrophoresis.
5. PCR amplification: mamK, two tubes; mamB, two tubes.
6. SDS page gel electrophoresis.
Person:Kaili Qin
Experiment:
1. Electrophoresis: mamK-PMD19-T vector1~15, gel is bad.
2. Pick bacteria: pick bacteria we want (mamK-PMD19-T vector1~10).
3. PCR: use PCR to detect mamK-pMD19-T vector1~10, 25ul system.
Person:Xue Sun
Experiment:
1. Recombination.
Week9
August 18th
Person:Yu Wang
Experiment:
1. Transformation.
2. Plate cultivation for 14h.
Person:Kaili Qin
Experiment:
1. Gel recovery: gel recovery of mamE.
2. Electrophoresis: gel is good for 8ng/ul each.
3. Bacterination: bacterination of E.coli with PSB1C3 plasmid for 3 tubes, 10ml each.
4. Bacterial coating: JQ11, QP11, KP1, BP1, BP2, LP21.
5. Gel recovery: gel recovery of PSB1C3 vector.
6. Electrophoresis: gel is good for 20ng/ul each.
Electrophoresis of PSB1C3 vector
Person:Qianyun Lu
Experiment:
1. PCR amplification: mamI, two tubes; mamQ, two tubes.
2. SDS page gel electrophoresis.
3. PCR production purification: RBS+mamQ, RBS+mamI.
4. SDS page gel electrophoresis.
5. PCR amplification: mamI, two tubes; mamJ, two tubes.
6. SDS page gel electrophoresis.
7. Transformation: JQ11, QP11, BP1, BP2, LP21, KP1.
8. SDS page gel electrophoresis.
9. PCR amplification: mamI, two tubes; mamB, two tubes.
10. SDS page gel electrophoresis: mamK; mamB.
lane6 mamK1; lane7 mamK2; lane8 mamB
Person:Wenjun Wang
Experiment:
1. Digestion: use enzyme EcoR I, Pst I to Cut PSB1C3 plasmid vector.
2. Digestion: use enzyme EcoR I, Pst I to Cut promoter J23106 and RBS B0032.
3. PCR: amplification mamE, mamJ sequence from PCR product.
4. Agarose electrophoresis: detect the plasmid PCR product.
5. Ligastion: promoter, RBS and mamQ.
6. Ligastion: mamQ and PSB1C3.
7. Ligastion: mamB and PSB1C3.
8. Ligastion: mamL and PSB1C3.
9. Ligastion: mamK and PSB1C3.
10. Transform: transform the ligastion product into Top 10.
August 19th
Person:Xue Sun
Experiment:
1. Pick bacteria and PCR examining.
Person:Kaili Qin
Experiment:
1. PCR: use PCR to detect mamK-PSB1C3 vector1~10,25ul system.
2. Electrophoresis: gel is bad.
Person:Qianyun Lu
Experiment:
1. Ligation: ligate the purified mamE with T vector.
2. PCR production purification: RBS+mamB, two tubes.
3. Pick the bacteria and culture the E.coli cells for four hours.
Person:Wenjun Wang
Experiment:
1. PCR: amplification mamE sequence from PCR product.
2. Agarose electrophoresis: detect the plasmid PCR product.
3. PCR: amplification mamI sequence from PCR product.
4. Product purification: purify the PCR product.
5. Agarose electrophoresis: detect the PCR product purification.
August 20th
Person:Qianyun Lu
Experiment:
1. Pick the bacteria and culture the E.coli cells for four hours.
2. PCR production purification: RBS+mamI, two tubes; RBS+mamB, one tube.
3. SDS page gel electrophoresis.
4. PCR amplification: mamI, four tubes.
5. SDS page gel electrophoresis.
6. Pick the bacteria and culture the E.coli cells for four hours.
Person:Qiu Wang
Experiment:
1. Sequencing J23101.
Person:Kaili Qin
Experiment:
1. PCR: use PCR to detect promoter-RBS-mamQ-PSB1C3 vector1~10, 25ul system; mamK - PSB1C31~28, 25ul system; mamE-PMD19-T vector1~8, 25ul system.
2. Electrophoresis: gel is bad.
August 21st
Person:Qianyun Lu
Experiment:
1. SDS page gel electrophoresis.
2. Miniprep: pSB1C3.
3. Digestion: three tubes are digested with XbaI and Pst-HF, two tubes are digested with EcoRI and Pst-HF.
4. SDS page gel electrophoresis.
Person:Kaili Qin
Experiment:
1. PCR: use PCR to clone mamL standard, mamB standard, mamQ standard, mamK standard.
2. Electrophoresis: mamL, K, B gel is good; mamQ, gel is bad.
Electrophoresis of production of mamK,B cloning
Person:Yu Wang
Experiment:
1. Bacterial activation and culturing, including control group and all experimental groups.
2. Preparing for measuring the fluorescence.
August 22th
Person:Wenjun Wang
Experiment:
1. PCR: Get mamQ sequence from PCR product and add prefix and suffix onto the squence.
2. Agarose electrophoresis: detect the plasmid PCR product.
3. Product purification: purify the PCR product.
Person:Qiu Wang
Experiment:
1. Bacterialculturing.
Person:Qianyun Lu
Experiment:
1. PCR amplification: mamB, four tubes; mamK, four tubes.
2. SDS page gel electrophoresis.
3. Gel extraction of DNA.
Person:Kaili Qin
Experiment:
1. PCR Purification: PCR Purification of mamL, K, B.
2. Enzyme digestion: enzyme digestion of mamL, K, B for 25ul system separately, use Xba1, Pst1.
August 23rd
Person:Xue Sun
Experiment:
1. Measuring the fluorescence.
Person:Kaili Qin
Experiment:
1. Electrophoresis: production of mamL, K, B enzyme digestion, gel is good.
2. Ligastion: mamL, K, B order pSB1C3 vector with chloramphenicol resistance.
3. Transform: transform the ligastion product into Top 10.
4. Bacterial coating.
Person:Qianyun Lu
Experiment:
1. Ligation: ligate RBS+ mamB with pSB1C3, ligate RBS+mamL and BOO15 with pSB1C3, ligate RBS+ mamK with pSB1C3.
2. PCR amplification: mamB, four tubes.
3. Transformation: L-P, B-P, K-P.
4. SDS page gel electrophoresis.
5. Ligation: ligate the purified mamC and mamE with T vector.
6. Transformation: C-T, E-T.
Person:Wenjun Wang
Experiment:
1. PCR: Get mamI sequence from PCR product and add prefix and suffix onto the sequence.
2. PCR: Get mamL sequence from PCR product and add prefix and suffix onto the sequence.
3. PCR: Get mamB sequence from PCR product and add prefix and suffix onto the sequence.
4. Agarose electrophoresis: detect the plasmid PCR product.
5. Product purification: purify the PCR product.
August 24th
Person:Wenjun Wang
Experiment:
1. PCR: amplification mamB+mamL, mamQ+mamI sequence from DNA ligastion product.
2. Agarose electrophoresis: detect the plasmid PCR product.
3. PCR: Get mamI sequence from AMB-1 bacterial strain.
4. PCR: Get mamI sequence from PCR product and add prefix and suffix onto the sequence.
5. Product purification: purify the PCR product.
6. Agarose electrophoresis: detect the plasmid PCR product.
Person:Qianyun Lu
Experiment:
1. Pick the bacteria and culture the E.coli cells for four hours.
2. PCR: C-T, E-T, L-P, B-P, K-P.
3. SDS page gel electrophoresis: PCR production.
Person:Kaili Qin
Experiment:
1. Pick bacteria: pick bacteria we want (mamL, K, B-PSB1C3).
2. PCR: use PCR to detect mamL, K, B-PSB1C3 vector1~10, 25ul.
3. Electrophoresis: gel is bad.
Person:Xue Sun
Experiment:
1. Result analysis.
Week10
August 25th
Person:Yu Wang
Experiment:
1. Bacterial culturing, including control group and all experimental group.
Person:Kaili Qin
Experiment:
1. Ligastion: mamC, mamE order pMD19-T vector.
2. Transform: transform the ligastion product into Top 10.
3. Bacterial coating.
Person:Qianyun Lu
Experiment:
1. PCR: L-P.
2. SDS page gel electrophoresis: mamC; mamE.
lane5: mamC, lane6: mamE
Person:Wenjun Wang
Experiment:
1. PCR: Get mamJ sequence from AMB-1 bacterial strain.
2. Digestion: use enzyme XbaI I, Spel I to cut mamI, mamL, mamB, mamQ.
3. Plasmid Extraction: extract the pSB1C3 plasmid which containing B0015 from E.coli.
4. Agarose electrophoresis: detect the plasmid extraction product and PCR product.
5. Ligastion: mamB and mamL; mamQ and mamI.
6. Agarose electrophoresis: detect the DNA digestion product.
7. Bacterial inoculation: inoculate the E.coli which contain pSB1C3 vector plasmid, C+.
8. PCR: amplification mamB+mamL, mamQ+mamI sequence from DNA ligastion product.
9. Agarose electrophoresis: detect the plasmid PCR product.
August 26th
Person:Xue Sun
Experiment:
1. Plasmid minprep.
2. Enzyme digestion and electrophoresis examining.
3. Recombination.
Person:Kaili Qin
Experiment:
1. Pick bacteria: pick bacteria we want (mamC, E-pMD19T- vector).
2. PCR: use PCR to detect mamC, E-pMD19T- vector1~10, 25ul.
3. Electrophoresis: gel is bad.
4. Pick bacteria: pick bacteria we want(mamC,E-PMD19T- vector ).
Person:Qianyun Lu
Experiment:
1. Pick the bacteria and culture the E.coli cells for four hours.
2. PCR: Q-P, K-P.
August 27th
Person:Qianyun Lu
Experiment:
1. Pick the bacteria and culture the E.coli cells for four hours: K-P.
2. PCR: Q-P, K-P.
Person:Qiu Wang
Experiment:
1. Transformation.
August 28th
Person:Qianyun Lu
Experiment:
1. PCR amplification: mamB, four tubes.
2. SDS page gel electrophoresis.
Person:Kaili Qin
Experiment:
1. PCR: use PCR to clone mamC standard.
2. PCR Purification: PCR Purification of mamL, K, B.
3. Electrophoresis: gel is good.
Person:Yu Wang
Experiment:
1. PCR amplification: mamB, four tubes.
2. SDS page gel electrophoresis.
August 29th
Person:Qiu Wang
Experiment:
1. Pick bacteria.
2. PCR.
3. Electrophoresis examining.
Person:Qianyun Lu
Experiment:
1. Digestion: digest RBS+mamK with XbaI and Pst-HF, digest RBS+mamB with XbaI and Pst-HF.
2. PCR amplification: mamE, three tubes; mamQ, three tubes, mamL, two tubes.
3. SDS page gel electrophoresis.
Person:Kaili Qin
Experiment:
1. PCR Purification: PCR Purification of mamE, L, Q.
2. Electrophoresis: gel is good.
August 30th
Person:Xue Sun
Experiment:
1. Pick bacteria, PCR and electrophoresis examining.
2. We find none of the fragment conforms to the aimed length, we explain it by the wrong proportion of part and plasmid backbone, so we prepared another experiment.
Person:Kaili Qin
Experiment:
1. PCR: use PCR to clone mamI standard.
2. Ligastion: mamL order pSB1C3 vector with chloramphenicol resistant.
3. Bacterination: bacterinate JB11 (promoter-RBS-PSB1C3), into 3 tubes of 10ml each.
Person:Qianyun Lu
Experiment:
1. SDS page gel electrophoresis.
2. Digestion: digest RBS+mamL with XbaI and Pst-HF, digest RBS+mamQ with XbaI and Pst-HF.
3. PCR amplification: mamQ, two tubes; mamK, two tubes.
August 31st
Person:Qianyun Lu
Experiment:
1. SDS page gel electrophoresis.
2. PCR amplification: mamB, two tubes; mamK, two tubes.
3. PCR production purification: RBS+mamB, two tubes.
4. Digestion: digest RBS+mamB with XbaI and Pst-HF.
Person:Kaili Qin
Experiment:
1. Plasmid miniprep: plasmid extraction of E.coli with JB11 (promoter-RBS-PSB1C3) plasmid for 6 tubes.
2. Enzyme digestion: enzyme digestion of JB11 (promoter-RBS-PSB1C3) for 25ul system, use Spe1, Pst1; enzyme digestion of mamQ for 25ul system, use Xba1, Pst1.enzyme digestion of mamB for 25ul system, use Xba1, Pst1.
3. PCR Purification: PCR Purification of mamC.
4. Electrophoresis: gel is good.
5. Ligastion: mamC, E order pMD19T-vector.
6. Transform: transform the ligastion product into Top 10.
7. Bacterial coating.
8. Ligastion：Promoter - RBS order mamQ, promoter-RBS order mamB.
Person:Xue Sun
Experiment:
1. Bacterial culturing, including control group and all experimental groups.
Week11
Septembert 1st
Person:Yu Wang
Experiment:
1. Plasmid minprep.
2. Enzyme digestion and electrophoresis examining.
3. Recombination.
Person:Kaili Qin
Experiment:
1. Pick bacteria: pick bacteria we want (mamL, pSB1C3 vector ).
2.Transform: transform promoter-RBS-mamB, promoter-RBS-mamQ, mamK-PSB1C3 into Top10.
3. PCR: use PCR to detect mamK-PSB1C3, 25ul system.
September 2nd
Person:Xue Sun
Experiment:
1. Transformation.
Person:Kaili Qin
Experiment:
1. Electrophoresis: mamL1~20, 3, 12, 14. gel is good.
Electrophoresis of mamL
2. Pick bacteria: pick bacteria we want (promoter-RBS-mamB, promoter-RBS-mamQ).
3. PCR: use PCR to detect promoter-RBS-mamB, promoter-RBS-mamQ.
September 3rd
Person:Qiu Wang
Experiment:
1. Grow bacteria in the culture plates.
Person:Kaili Qin
Experiment:
1. Bacterial coating: mamK-PSB1C3.
September 4th
Person:Kaili Qin
Experiment:
1. Pick bacteria: pick bacteria we want (mamK-PSB1C3).
2. PCR: use PCR to detect (mamK-PSB1C3).
3. Electrophoresis: mamK-PSB1C3 number5, gel is good, others are bad.
Person:Yu Wang
Experiment:
1. Pick up the single colony, PCR, run gel and sequencing.
2. Miniprep of B0015 and four experiment groups.
3. Cultured cells of PSB1C3 for making the backbone.
September 5th
Person:Qiu Wang
Experiment:
1. Miniprep the PSB1C3.
2. Enzyme digestion: B0015 with EcoR I and SpeI; the experiment group 1 use XbaI and PstI; the other experiments group use EcoR I and XbaI; the PSB1C3 with EcoRI and PstI.
3. Gel extraction PSB1C3 and the experiment group 1.
4. Recombination.
Person:Kaili Qin
Experiment:
1. Pick bacteria: pick bacteria we want (mamK-PSB1C3).
2. PCR: use PCR to detect (mamK-PSB1C3).
3. Electrophoresis: gel is bad.
September 6th
Person:Xue Sun
Experiment:
1. Transformation.
Week12
September 9th
Person:Yu Wang
Experiment:
1. Pick up single colony, PCR, and sequencing.
Person:Kaili Qin
Experiment:
1. PCR: use PCR to clone mamC.
2. Electrophoresis: gel is bad.
3. Enzyme digestion: enzyme digestion of mamI, mamQ; 25ul system, use Xba1, Pst1.
4. Electrophoresis: gel is good.
September 10th
Person:Xue Sun
Experiment:
1. Culture the experiment group 1.
Person:Kaili Qin
Experiment:
1. Pick bacteria: pick bacteria we want (mamQ1~40, mamI1~10).
2. PCR: use PCR to detect mamQ, mamI.
3. Electrophoresis: gel is good.
Electrophoresis of mamI
September 11st
Person:Qiu Wang
Experiment:
1. Miniprep and enzyme digestion with EcoR I and XbaI.
2. Gel extraction.
September 12th
Person:Kaili Qin
Experiment:
1. Bacterination: Bacterinate E.coli with pSB2K3-RFP into tubes of 10ml.
Person:Xiaodong Zhong
Experiment:
1. TEM (Electron microscopy) of Magnetospirillum Magneticum AMB-1.
September 13th
Person:Kaili Qin
Experiment:
1. Plasmid miniprep: plasmid extraction of pSB2K3 for 5 tubes.
2. Enzyme digestion: enzyme digestion of pSB2K3 for 50ul system, use EcoR1, Pst1.
3. Gel recovery: gel recovery of the product of enzyme digestion.
4. Electrophoresis: gel is good.
September 14th
Person:Xue Sun
Experiment:
1. Inoculate the cell to culture plates, containing the control group, experiment 3 and experiment 4.
Week13
September 15th
Person:Yu Wang
Experiment:
1. Fluorescence detection.
Person:Qianyun Lu
Experiment:
1. Microscopy of Magnetospirillum magneticum AMB-1.
Person:Xiaodong Zhong
Experiment:
1. Magnetic analysis: detect the magnetism of Magnetospirillum magneticum AMB-1.
September 20th
Person:Kaili Qin
Experiment:
1. Plasmid miniprep: plasmid Extraction of double expression plasmid for 9 tubes.
2. Electrophoresis: gel is good.
Electrophoresis of double expression plasmid
