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Revision as of 14:27, 4 September 2013

Journal

July

Week 3

• Introduction given by our lab instructors
• General techniques: plasmid/PCR purification, inoculation, gel extraction, restriction & ligation.
• Safety and waste disposal training
• Introduction to CloneManager
• General preparations: sterile mQ, sterile eppendorf

August

Week 1

• Experiment 1
• Inoculate E.Coli DH5a + pTGD-ccdA-Pmb1GFP-CmFRT
• Purify plasmid pTGD-ccdA-Pmb1GFP-CmFRT using Qiagen spin mini kit: Nanodrop -- 362.4 ng/µL
• Preparative Restriction Digest (RD) of purified plasmid: BspHI & BstAPI: expected fragments of 5541bp and 903 bp
• Gel purify RD-fragment of 5541 bp using Qiagen Qiaquick gel extraction kit: Nanodrop -- 70.4 ng/µL
• PCR on RD (HR-ccdA-Pmb1GFP-HR)and on plasmid pTGD-ccdA-PMbAFGP-CmFRT with MDM588 & MDM589
  ->HiFi PCR using Primestar polymerase. Analytical gel: negative
  ->PCR using Q5 polymerase. Analytical gel: negative
  ->PCR using Roche. Analytical gel: negative
  ->Touchdown PCR using Q5 polymerase. Analytical gel: negative
• Control plasmid pTGD-ccdA-PMbAFGP-CmFRT: RD with AclI. Expected fragments: 758 bp, 1730 bp and 3956 bp. Analytical gel: negative --> Problem with plasmid pTGD-ccdA-PMbAFGP-CmFRT
• Transformation: Knock in with lineair back-up DNA by electroporation. Incubation of the transformed cells and transfer the culture on a Cm plate.
• Colony PCR on 48 colonies using crimson taq polymerase with two primer pairs: MDM0141/MDM0010 and MDM0046/MDM123. Expected fragments: 550bp & 2450 bp. Analytic gel: 4 positives
• Colony PCR on 4 positive and 4 negative colonies using crimson taq polymerase with out primers: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: negative .
• 2 Colony PCR on 4 positive and 4 negative colonies using Taq and Phire polymerase with out primers: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: negative .
• Colony PCR on 4 positive and 4 negative colonies using Emerald polymerase with out primers: MDM0046/MDM0010. Expected fragment: ca. 5500 bp. Analytic gel: 1 positive: colonie 35 .


• Experiment 2
• Inoculate E. coli DH5a + p5SpFRT-T7ccdB
  Inoculate E. coli DH5a + p10SpFRT-T7ccdB
  Inoculate E. coli DH5a + p20SpFRT-T7ccdB
• Purify plasmids using Qiagen spin mini kit: nanodrop
  p5SpFRT-T7ccdB: 119,6 ng/µl
  p10SpFRT-T7ccdB: 156,3 ng/µl
  p20SpFRT-T7ccdB: 392,9 ng/µl
• CcdB operon:
  -> HiFi PCR of plasmids p5SpFRT-T7ccdB, p10SpFRT-T7ccdB and p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB operon
  -> Purification of CcdB operon PCR fragment using Qiagen Qiaquick PCR purification kit and checked on analytical gel: nanodrop
  p5: 174,6 ng/µl
  p10: 24,5 ng/µl (consistent with small band on the analytival gel)
  p20: 104,3 ng/µl
  -> Redo of inoculation E. coli DH5a + p5SpFRT-T7ccdB, E. coli DH5a + p10SpFRT-T7ccdB and E. coli DH5a + p20SpFRT-T7ccdB
  -> Redo of purification of plasmids p5SpFRT-T7ccdB, p10SpFRT-T7ccdB and p20SpFRT-T7ccdB using Qiagen Spin minikit: nanodrop
  p5SpFRT-T7ccdB: 21,1 ng/µl
  p10SpFRT-T7ccdB: 25,1 ng/µl
  p20SpFRT-T7ccdB: 24,6 ng/µl
• Vectors pSB4A5 (plate 5, well 5I), pSB3T5 (plate 2, well 8D) and pSB6A1 (plate 2, well 2L):
  -> Resuspend plasmids from the iGEM kit
  -> Transform in E.Coli Top10 subcloning cells using elektroporation
  -> Plate pSB4A5 and pSB6A1 on ampicillin plate and pSB3T5 on tetracyclin and grow overnight at 37°C
  -> Plates with transformants: pSB6A1 lots of colonies, pSB3T5 sufficient colonies and pSB4A5 no colonies
• Vectors pSB4A5 (plate 5, well 5I):
  -> Resuspend plasmids from the iGEM kit
  -> Transform in E.Coli Top10 subcloning cells using elektroporation
  -> Plate on ampicillin plate and grow overnight at 37°C (1 plate 150 µl, 1 plate 50 µl)
  -> Plates with transformants: pSB4A5 no colonies
• Inoculation colonies of pSB3T5 and pSB6A1 -> at the end of the day replaced from 37°C to 30°C
• Vector pSB4A5 (plate 5, well 1I and plate 2, well 2J as backup) and pSB6A1 (plate 5, well 1K as backup):
  -> Resuspend plasmid pSB4A5 and pSB6A1 from the iGEM kit
  -> Transform in E. coli Top10 subcloning cells (heat shock)
  -> Plate transformation on ampicillin plate and grow overnight at 37°C (3 plates: 10^-2,10^-1 and 10⁰)
  -> Plates with transformants: pSB4A5 (plate 2, well 2J) colonies, pSB4A5 (plate 5, well 1I) no colonies and pSB6A1 (plate 2, 2L) few colonies
• Inoculate pSB3T5 and pSB6A1 again and in warm chamber (37°C).
• Inoculation colonies of pSB4A5 (plate 2, well 2J) and pSB6A1 (plate 2, well 2L) (2 times: one for further work and one for cryovials)
• Purification of plasmids pSB3T5, pSB6A1 and pSB4A5
• Generation of restriction digest fragments of T7-ccdB insert (from p5SpFRT-T7ccdB and p20SpFRT-T7ccdB) and vector (pSB3T5 (2x), pSB4A5 (from plate 2, well 2J) and pSB6A1 (1x from plate 5, well 1K and 1x from plate 2, well 2L) with XbaI and PstI-HF (gel purificy RD-fragments using Qiagen Qiaquick gel extraction kit): Nanodrop
  p5: 43,7 ng/µl
  p20: 24,2 ng/µl
  3T5 (inoculation 1): 23,5 ng/µl
  3T5 (inoculation 2): 23, ng/µl
  4A5: 40,5 ng/µl
  6A1 (plate 2, 2L): 40,1 ng/µl
  6A1 (plate 5, 1K): 30,4 ng/µl
• Ligation of T7ccdB from p5SpFRT-T7ccdB and pSB6A1 (1x from plate 5, well 1K and 1x from plate 2, well 2L), p20SpFRT-T7ccdB and pSB3T5, p20SpFRT-T7ccdB and pSB4A5 (from plate 2, well 2J) (3(insert)/1(plasmid) ratio)
• Transformation in E.coli Top10 (heat shock)
• Plate transformation on ampicillin (pSB4A5 and pSB6A1) and tetracycline plate (pSB3T5) and grow overnight at 37°C (2 plates: 10^-2, 10^-1)
• New transformation in E.coli Top10 from pSB4A5 and pSB6A1 using heat shock
  -> Plates with transformants: no colonies
• New ligation with Rd-fragments created before(vectors pSB3T5, pSB4A5, pSB6A1 (plate 5, well 1K and insert T7-ccdB created in experiment 6), at 22,5°C o/n)
• CcdB operon:
  -> HiFi PCR of plasmids p5SpFRT-T7ccdB, p10SpFRT-T7ccdB and p20SpFRT-T7ccdB with MDM0586 & MDM0587 to amplify ccdB operon


• Experiment 6
• CcdB operon: HiFi PCR of plasmids p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB operon
  nanodrop: 301,5 ng/µl
• pSB1C3
  -> Inoculate E. coli DH5a + pSB1C3
  -> Purify plasmids using Qiagen Qiaprep Spin minikit: nanodrop: 200,2 ng/µl
• Restriction of CcdB operon and pSB1C3 with XbaI and PstI
  Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
  RD_T7-ccdB: 16.9 ng/µl
  RD_pSB1C3: 12.3 ng/µl
• Ligation of CcdB operon and pSB1C3
• Transformation in E.Coli Top10 subcloning cells using heat shock
  and incubation on chloramphenicol agar plates at 37°C: negative

Week 2

• Experiment 1


• Experiment 2


• Experiment 6
• Again transformation in E.Coli Top10 subcloning cells using heat shock
  and incubation on chloramphenicol agar plates: negative
• CcdB operon: HiFi PCR of plasmids p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB operon
• Again restriction of CcdB operon and pSB1C3 with XbaI and PstI
  Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
  RD_T7-ccdB: 16.6 ng/µl
  RD_pSB1C3: 17.7 ng/µl
• Ligation of CcdB operon and pSB1C3: overnight at 16°C
• Transformation in E. coli DH5a using heat shock
  and incubation on chloramphenicol agar plates + glucose at 30°C: negative
• Restriction of CcdB operon and pSB1C3 with XbaI and PstI, using DpnI
• Control of restriction fragments: positive, so we hope that ligation will succeed
• Ligation of CcdB operon and pSB1C3: overnight at 16°C
• Transformation in E. coli DH5a
  and incubation on chloramphenicol agar plates: negative

Week 3

• Experiment 1


• Experiment 2


• Experiment 6
• Again transformation in E. coli DH5a
  (because we are shore that the fragments were present for ligation and so will be succeed)
  and incubation on chloramphenicol agar plates: negative
• Control of ligations (with PCR using primers MDM0606 and MDM0607): negative
• Ligation of CcdB operon and pSB1C3: 15 minutes at 16°C
• Again transformation in E. coli DH5a
  and incubation on chloramphenicol agar plates: negative
• CcdB operon: HiFi PCR of plasmids p20SpFRT-T7ccdB with MDM0586/MDM0587 to amplify ccdB operon
• Again restriction of CcdB operon and pSB1C3 with XbaI and PstI
  Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
  RD_T7-ccdB: 15.3 ng/µl
  RD_pSB1C3: 16.8 ng/µl
• Again ligation of CcdB operon and pSB1C3: 1 hour at room temperature
• Transformation in E. coli DH5a
  and incubation on chloramphenicol agar plates: positive
• Colony PCR of colonies: negative
• Designing primers for Gibson Assembly

Week 4

• Experiment 2
• Q5 PCR of CcdB operon and plasmids (pSB3T5 and pSB4A5), using the designed Gibson primers
  Purification of PCR-product after using DpnI
  Control of fragments on a gel => Backbones are present, CcdB operons not
• PrimeStar PCR of CcdB operons, using the designed Gibson primers
  Purification of PCR-product after using DpnI
  Control of fragments on a gel => CcdB operons are still not present
• Q5 PCR of CcdB operons on a linear CcdB operon, using the designed Gibson primers
  Purification of PCR-product
  Control of fragments on a gel => CcdB operon is visible
• Gibson Assembly (1h at 50°C)
• Transformation in E. coli DH5a using heat shock (42°C)
  and plate pSB4A5 on an ampicillin plate and pSB3T5 on tetracyclin plate at 37°C: positive
  Strange phenomena: most colonies are red, let’s control it
• Colony PCR of colonies (derived from Gibson Assembly): negative


• Experiment 3


• Experiment 6
• While waiting for the Gibson Assembly primers
• Again restriction of CcdB operon and pSB1C3 using XbaI and PstI
  Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
  RD_T7-ccdB: 10.5 ng/µl
  RD_pSB1C3: 7.2 ng/µl
• Ligation of CcdB operon and pSB1C3: overnight at 16°C
• Transformation in E. coli DH5a using heat shock
  and incubation on chloramphenicol agar plates + glucose at 30°C: negative
  
  
• After receiving the primers for Gibson Assembly
• Q5 PCR of CcdB operon and pSB1C3, using the designed Gibson primers
  Purification of PCR-product after using DpnI
  Control of fragments on a gel => Backbone is present, CcdB operon not
• PrimeStar PCR of CcdB operon, using the designed Gibson primers
  Purification of PCR-product after using DpnI
  Control of fragments on a gel => CcdB operon is not present
• Q5 PCR of CcdB operon on a linear CcdB operon, using the designed Gibson primers
  Purification of PCR-product
  Control of fragments on a gel => CcdB operon is visible
• Gibson Assembly (1h at 50°C)
• Transformation in E. coli DH5a using heat shock (42°C)
  and incubation on chloramphenicol agar plates at 37°C: positive
  Strange phenomena: colonies are red, let’s control it
• Colony PCR of colonies (derived from Gibson Assembly): negative

September

Week 1

• Experiment 2


• Experiment 4


• Experiment 6

Days: Hours: Mins: Secs: Until Jamboree Lyon Tweets van @iGEM_UGent