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 (expected fragment of 2300 bp): 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 purify RD-fragments using Qiagen Qiaquick gel extraction kit (expected fragment of 2157 bp for T7-ccdB, 3229 bp for pSB3T5, 3372 bp for pSB4A5 and 3999 bp for pSB6A1): 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)
-> Plates with transformants: no colonies
New transformation in E.coli Top10 from pSB4A5 and pSB6A1 using heat shock
-> Plates with transformants: pSB6A1 2 colonies, pSB4A5 few 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:
Restriction Digest of T7-ccdB: 16.9 ng/µl
Restriction Digest of 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
cPCR with Taq polymerase of colonies pSB6A1 (primers: MDM0096 and CLG0019) and pSB4A5 (primers: MDM0095 and CLG0019): negative
New transformation with pSB3T5, pSB4A5 and pSB6A1 using heat shock in E.coliDH5a -> Plate on normal plates and glucose plates (100 ml 2% glucose, to avoid leaky expressing of ccdB)
-> Plates with transformants: pSB6A1 some colonies, pSB4A5 some colonies and pSB3T5 no colonies
Gel elektroforesis on RD-fragments from plasmids: show expected fragments
cPCR on the pSB4A5 (primers: MDM0095 and CLG0019) and pSB6A1 (primers: MDM0096 and CLG0019) colonies with Taq polymerase: one positive colony of pSB6A1-T7ccdB (fragment of 437 bp in lane 14)
inoculation of positive pSB6A1-T7ccdB colony from back up plate (3x: one for sequencing, 1 for cryovial and 1 for experiment 3)
Plate transformation mixtures again on glucose plates and grow overnight at 30°C
->Plates with transformants: pSB4A5 colonies, pSB3T5 no colonies
cPCR on pSB4A5 colonies (primers: MDM0095 and CLG0019) with Taq polymerase: negative
New ligation with rest of RD-fragments of plasmids pSB3T5 (2 times) and pSB4A5 and ccdB from experiment 6 and transformation in DH5a using heat shock
Due to lack of success: start from the beginning.
Inoculation of pSB3T5, pSB4A5, p5SpFRT-T7ccdB, p10SpFRT-T7ccdB and p10SpFRT-T7ccdB from cryovials.
Also resuspend pSB3T5 (plate 5, well 7C) from iGEM kit, transform in E.coli DH5a using heat shock, plate on tetracycline plates and incubate overnight at 37°C
-> no colonies
-> transform again in DH5a using heat shock
-> again no colonies
Purification of plasmids using Qiagen spin minikit: concentrations deduced from gel
p5SpFRT-T7ccdB: +/- 40 ng/µl
p10SpFRT-T7ccdB: +/- 40 ng/µl
p20SpFRT-T7ccdB: +/- 40 ng/µl
pSB3T4: +/- 250 ng/µl
pSB4A5 (1): +/- 400 ng/µl
pSB4A5 (2): +/- 350 ng/µl
PCR on ccdB operon using Q5 polymerase (primers: MDM0586_Fw-Trc-ccdB-G00000 and MDM0587_Rv-Trc-ccdB-G00001)
cPCR on pSB3T5 colonies from old plates using Taq polymerase (primers: MDM0602 and CLG0019, expected fragment of 503 bp): negative
Restriction of ccdB operon, pSB3T5 and pSB4A5 using PstI-HF and XbaI (gel purify RD-fragments using Qiagen Qiaquick gel extraction kit (expected fragment of 2157 bp for T7-ccdB, 3229 bp for pSB3T5 and 3372 bp for pSB4A5): concentrations deduced from gel
Ligation of pSB3T5 and T7ccdB (from p5SpFRT-T7ccdB), pSB4A5 and T7ccdB (from p5SpFRT-T7ccdB) and pSB4A5 and T7ccdB (from p20SpFRT-T7ccdB) with T4 DNA ligase at 16°C overnight
Transformation of ligation mixtures in DH5a using heat shock
-> no colonies
Experiment 6
Again transformation in E.Coli Top10 subcloning cells, using heat shock (42°C),
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:
Restriction Digest of T7-ccdB: 16.6 ng/µl
Restriction Digest of pSB1C3: 17.7 ng/µl
Ligation of CcdB operon and pSB1C3: overnight at 16°C
Transformation in E. coli DH5a, using heat shock (42°C),
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, using heat shock (42°C),
and incubation on chloramphenicol agar plates: negative
Week 3
Experiment 1
Experiment 2
pSB6A1-T7ccdB sent for sequencing with primers MDM0096 (A471969), MDM0060 (A471682) and MDM0039 (A471681)
-> correct sequence
Transform ligation mixtures again in DH5a, using heat shock (42°C),
-> no colonies
PCR on ccdB operon with Q5 polymerase (primers: MDM0586_Fw-Trc-ccdB-G00000 & MDM0587_Rv-Trc-ccdB-G00001) and PCR purification using Qiagen Qiaquick PCR purification kit: nanodrop
p5: 15.2 ng/µl
p10: 18.6 ng/µl
p20: 43.5 ng/µl
Inoculation of pSB3T5 and pSB4A5 from cryovials and plasmid purification using Qiagen Spin minikit: nanodrop
pSB3T5: 75.7 ng/µl
pSB4A5: 190.0 ng/µl
Restriction of ccdB operon, pSB3T5, pSB4A5 and the pSB6A1-T7ccdB transformant with PstI-HF and XbaI (gel purify RD-fragments using Qiagen Qiaquick gel extraction kit (expected fragment of 2157 bp for T7-ccdB, 3229 bp for pSB3T5 and 3372 bp for pSB4A5)
Ligation
Transformation in E.coli DH5a
-> no colonies
-> plate transformants on plates with a lower antibiotic concentration (50% lower)
-> no colonies
Design primers for Gibson assembly
Experiment 6
Again transformation in E. coli DH5a (because we are sure 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:
Restriction Digest of T7-ccdB: 15.3 ng/µl
Restriction Digest of pSB1C3: 16.8 ng/µl
Again ligation of CcdB operon and pSB1C3: 1 hour at room temperature
Transformation in E. coli DH5a, using heat shock (42°C),
and incubation on chloramphenicol agar plates: positive
Colony PCR of colonies: negative
Designing primers for Gibson Assembly
Week 4
Experiment 2
Inoculation of pSB3T5, pSB4A5, pSB6A1-T7ccdB, p5SpFRT-T7ccdB, p10pFRT-T7ccdB and p20pFRT-T7ccdB and plasmid purification using Qiagen Spin minikit: nanodrop
PCR on ccdB operon using Q5 polymerase (primers: MDM0586_Fw-Trc-ccdB-G00000 and MDM0587_Rv-Trc-ccdB-G00001)
Restriction of pSB3T5, pSB4A5 and ccdB operon with PstI-HF and XbaI (gel purify RD-fragments using Qiagen Qiaquick gel extraction kit (expected fragment of 2157 bp for T7-ccdB, 3229 bp for pSB3T5 and 3372 bp for pSB4A5): nanodrop:
-> pSB3T5: 2,7 ng/µl
-> pSB4A5: 7,8 ng/µl
Q5 PCR of CcdB operon and plasmids (pSB3T5 and pSB4A5), using the designed Gibson primers
Purification of PCR-product,using Qiagen Qiaquick PCR purification kit, after DpnI treatment
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,using Qiagen Qiaquick PCR purification kit, after DpnI treatment
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:
Restriction Digest of T7-ccdB: 10.5 ng/µl
Restriction Digest of pSB1C3: 7.2 ng/µl
Ligation of CcdB operon and pSB1C3: overnight at 16°C
Transformation in E. coli DH5a, using heat shock (42°C),
and incubation on chloramphenicol agar plates at 37°C: positive
Colony PCR (Taq) of these colonies: 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, using Qiagen Qiaquick PCR purification kit, 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, using Qiagen Qiaquick PCR purification kit
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 (Taq) of colonies (derived from Gibson Assembly): negative
September
Week 1
Experiment 2
Restriction of CcdB operon and pSB3T5 using EcoRI and SpeI
Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
RD_T7-ccdB: 14.4 ng/µl
RD_pSB3T5 : 7.2 ng/µl
Restriction of CcdB operon and pSB4A5 using EcoRI and SpeI
Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
RD_T7-ccdB: 14.4 ng/µl
RD_pSB4A5: 7.8 ng/µl
Ligation of CcdB operon and pSB3T5 and of CcdB operon and pSB4A5: 25 minutes at room temperature
Transformation in E. coli DH5a using heat shock
and incubation of pSB3T5 on tetracycline agar plates and of pSB4A5 on ampicillin agar plates at 37°C: negative
Experiment 4
Experiment 6
Restriction of CcdB operon and pSB1C3 using EcoRI and SpeI
Gel purify fragments using Qiagen Qiaquick gel extraction kit: nanodrop:
RD_T7-ccdB: 14.4 ng/µl
RD_pSB1C3: 4.4 ng/µl
Ligation of CcdB operon and pSB1C3: 25 minutes at room temperature
Transformation in E. coli DH5a using heat shock
and incubation on chloramphenicol agar plates at 37°C: negative
Q5 PCR of pSB1C3, using the designed Gibson primers and the linearised plasmid
Purification of PCR-product, using Qiagen Qiaquick PCR purification kit: nanodrop:
pSB1C3-backbone: 109.9 ng/µl
Gibson Assembly (1h at 50°C)
Transformation in E. coli DH5a using electroporation (Time Constant: 4.4)
and incubation on chloramphenicol agar plates at 37°C: positive
Colony PCR (Crimson) of colonies:
6 colonies can possible be positive (but it is not sure)
Control of fragment of pSB1C3 on a gel => Backbone is not present
Inoculate the 6 colonies (overnight)
Plasmid purification of the 6 colonies + restriction to control of they are the good colonies (on a gel)
There is a possibility that they are the good ons, so we send it to sequenate
Q5 PCR of pSB1C3, using the designed Gibson primers and the linearised plasmid
Purification of PCR-product using Qiagen Qiaquick PCR purification kit:
nanodrop: pSB1C3-backbone: 58.0 ng/µl
Control of fragment of pSB1C3 on a gel => Backbone is not present
Q5 PCR of pSB1C3, using the designed Gibson primers and the restriction digest of pSB1C3
Purification of PCR-product using Qiagen Qiaquick PCR purification kit:
nanodrop: pSB1C3-backbone: 76.2 ng/µl
Control of fragment of pSB1C3 on a gel => Backbone is present
Gibson Assembly (1h at 50°C)
Transformation in E. coli DH5a, using electroporation (Time Constant: 4.3)
and incubation on chloramphenicol agar plates at 37°C: positive => many colonies
Colony PCR (Crimson) of colonies:
we tested 48 colonies and 4 colonies show the right fragment
Inoculate the 4 good colonies
Plasmid purification of the 4 colonies + restriction to control of they are the good colonies (on a gel)
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