Team:Freiburg/Labjournals/Cloning/September

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2015/09/02

Digest: pOP, pILS3 and K592009 with EcoRI (LS)

amountingredient
~5µlplasmid
5µlCut Smart
1µlEcoRI
up to 50µldH2O
  • incubation at 37°C, ~1h

PCR Purification of EcoRI digest (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
concentration [ng/µl]
pOP 3.3
pILS3 23.
BBa_K592009 2.3

Digest of pOP, pILS3 and BBa_K592009 with PstI (JN)

ingredient amount [µl]
DNA16/17/18
PstI1
buffer 3.15
dH2O28/27/26
  • 37°C for 1h
  • analysis on 1% agarose gel
Expected fragment sizes: pOP ~5000bp, BBa_I13504 ~700bp, BBa_K592009 ~700bp

Gel-ex of BBa_I13504 (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
  • concentration: 10.2ng/µl

Repetition of pOP Digest with EcoRI (JN)

ingredient amount [µl]
DNA5
EcoRI1
CutSmart5
dH2O39
  • 37°C for 1h

PCR Purification of pOP Digest

  • Qiagen kit
  • eluted in 20µl dH20
  • concentration: 3.7ng/µl

pOP digest with PstI

ingredient amount [µl]
DNA17
PstI1
buffer 3.15
dH2O27
  • 37°C for 1h
  • analysis on 1% agarose gel
Expected fragment size: ~5000bp

Gel-ex of pOP (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
  • concentration: 6.4ng/µl

Ligation of pOP+BBa_I13504 (JN)

  • Backbone: 3.4 µl
  • BBa_I13504: 10.5 µl
  • buffer: 2 µl
  • T4 ligase: 1 µl
  • dH2O: 3.1 µl

Trafo: Ligation of pOP BBA_I13504 in E.coli T10 (LS)

  • 7 µl of ligation
  • transformation according to protocol

2015/09/03

Inoculation of pOP_BBa_I13504 (LS)

  • picked 3 clones
  • inoculation in LB-Amp (5ml)
  • incubation at 37°C

Inoculation of pOP_BBa_I13504 (JN)

  • 10 more clones were picked and inoculated o/n
  • each clone in 5ml of LB-Amp

2015/09/04

Mini-Prep of pOP_Anderson_GFP (JN)

  • all liquid cultures were fluorescent, only three were prepped, four more were pelleted in stored in the freezer as backup
  • Qiagen kit
  • eluted in 30µl dH2O
concentration [ng/µl]
pOP_Anderson_GFP 4 21.1
pOP_Anderson_GFP 9 42.9
pOP_Anderson_GFP 11 37.2
  • sample 9 and 11 were sent in for sequencing

Inoculation of pOP (JN)

  • inoculation of 3 colonies from already performed re-trafo (LS)
  • 3 liquid cultures with 5ml LB-Amp medium each
  • inoculated o/n

2015/09/05

Miniprep: pOP (LS)

  • 15µl of pOP
  • Zymo kit
  • elution in 65µl: 81,0 ng/µl

Digest: pOP and K592009 with EcoRI (LS)

amountingredient
10µl/~4µlpOP/K592009(all that was left)
1µlEcoRI
5µlCut Smart
up to 50µldH20
  • incubation at 37°C for 1h

Clean-up of Digest (LS)

  • Zymo PCR clean-up kit
  • elution in 20µl:
  • pOP:21,3ng/µl
  • K592009:2,7ng/µl

Digest: pOP and K592009 from clean-up with PstI (LS)

amountingredient
18µlpOP/K592009
5µlbuffer 3.1
1µlPstI
36µl dH2O
  • incubation at 37°C, 1h
  • analysis on 1% agarose gel:

-small band visible for pOP -nothing for K592009

Digest of pOP and K592009 ith EcoRI an dPstI. Expected badn lenght for pOP: ~5500bp and for K592009 ~700bp. Digest worked for pOP, but not for K592009.

Inoculation: 3x 5ml LB-Cml wit K592009 (LS)

  • incubation at 37°C, o/n

2015/09/06

Mini-Prep of K592009 (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
177.0 ng/µl
294.4 ng/µl
364.6 ng/µl

Digest of all three preps with EcoRI (JN)

amountingredient
5µlK592009
1µlEcoRI
5µlCut Smart
39µldH20
  • incubation at 37°C for 1h

PCR clean-up (JN)

  • Qiagen kit
  • eluted in 20 µl dH2O
17.6 ng/µl
29.2 ng/µl
37.2 ng/µl

Digest: K592009 with PstI (JN)

amountingredient
25µlDNA
1µlPstI
5µlbuffer 3.1
19µldH20
  • incubation at 37°C for 1h
  • analysis on 1% agarose gel

Gel-ex: Digest of K592009 and pOP (LS)

  • qiagen kit
  • eluted in 20µl
  • pOP: 3,9 ng/µl
  • K59209: 9,0 ng/µl

Ligation: K592009 into pOP (LS)

ingredientamount [µl]
pOP12,83
K5920092,33
T4 ligase buffer2
T4 ligase1
dH2o2
  • incubation at Rt for 1h

Transformation of ligation into E.coli T10 (LS)

  • 7µl of ligation mix
  • transformation according to protocol
  • plated on LB-Amp
  • incubation at 37°C o/n

2015/09/10

Clean-up of pOP EcoRI digest (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
  • concentration: 1.8ng/µl

Subsequent digest with PstI (JN)

ingredient amount [µl]
DNA16
PstI1
buffer 3.15
dH2O28
  • 37°C for 1h
  • analysis on 1% agarose gel, see below

Digest of pOP_Anderson_GFP with EcoRI (JN)

ingredient amount [µl]
DNA5
EcoRI1
CutSmart2
dH2O12
  • 37°C for 1h

Clean-up of pOP_A_GFP EcoRI digest (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
  • concentration: 28.0 ng/µl

Subsequent digest with PstI (JN)

ingredient amount [µl]
DNA16
PstI1
buffer 3.15
dH2O28
  • 37°C for 1h
  • analysis on 1% agarose gel

Gel-ex of pOP digested with EcoRI and PstI (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
  • concentration: 9.6 ng/µl

2015/09/11

Liquid cultures of Ligation pOP + BBa_K592009 (JN)

  • 10 colonies were picked
  • inoculated in 5ml LB-Amp each
  • 37°C, shaking, until evening

OD of liquid cultures (JN)

  • OD of the liquid cultures from the morning was measured
  • cultures were diluted to a OD of 0.4 in a volume of 5ml and grown again for half an hour
  • induction with 0.5µl of IPTG
  • incubated o/n shaking at 37°C

2015/09/12

Mini-Prep of 3 induced cultures of pOP_K592009 (JN)

  • unfortunately, no liquid were blue as they should be if expressing the protein
  • Zymo Research kit
  • eluted in 30µl dH2O
concentration [ng/µl]
pOP_K592009 148.7
pOP_K592009 446.5
pOP_K592009 752.7

Test-digest of pOP_K592009 with EcoRI (JN)

ingredient amount [µl]
DNA15
EcoRI1
CutSmart2
dH2O2
  • 37°C for 1h

Clean-up of pOP_K592009 EcoRI digest (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
concentration [ng/µl]
pOP_K592009 120.0
pOP_K592009 414.0
pOP_K592009 713.7

Subsequent digest with PstI (JN)

ingredient amount [µl]
DNA16
PstI1
buffer 3.12
dH2O1
  • 37°C for 1h
  • analysis on 1% agarose gel, band fot the backbone was clearly visible at the right height but no band for the insert was visible

Digest: BBa_I13504 with EcoRI (LS)

ingredientsvolume
BBa_I1350410µl
CutSmart4µl
EcoRI1µl
dH2O25µl
  • incubation at 37°C for 1h
  • clean-up with PCR-Clean-up kit (Roche)
  • eluted in 20µl: 34,5ng/µl

Digest: Clean-up (BBa_I13504) with PstI (LS)

ingredientsvolumes
BBa_I13504 (EcoRI digested)
buffer 3.14µl
PstI1µl
dH2017µl
  • incubation at 37°C for 1h
  • analysis on 1% agarose gel, correct band of the BioBrick was cut out of the gel

Gel-ex of BBa_I13504 (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
  • concentration: 9.1ng/µl

Ligation of pOP + BBa_I13504 (JN)

ingredientsvolumes
pOP7µl
BBa_I135042.9µl
buffer2µl
T4ligase1µl
dH207.1µl
  • 1h at RT

Trafo of pOP_I13504 into BL21 (LS)

  • 7 µl of ligation mix
  • transformation according to protocol
  • plated on LB-Amp

2015/09/13

Inoculation of pOP_I13504 (LS)

  • inoculation of 10 x 5 ml LB-Amp with ligation clones
  • incubation at 37°C

OD Measurement of liquid cultures (JN)

  • OD for each liquid culture was measured
  • dilution for each culture to a final OD of 0.4
  • incubation at 37°C for 30min, shaking
  • OD was measured again –> for all cultures about 0.5
  • induction with 5µl IPTG for a final concentration of 1mM
  • incubation at 37°C, shaking

Checked for fluorescence (JN/LS)

  • measuring of fluorescence in all 10 cultures with UV light
  • all cultures showed fluorescence

Restreaked 3 cultures of pOP_I13504 (LS)

  • incubation at 37°C o/n

2015/09/14

Inoculation of pOP_I13504 (LS)

  • 6 x 5ml LB-Amp, two cultures of each colony
  • incubation at 37°C, shaking

OD measurement + induction with IPTG o/n (JN)

  • cultures were diluted to obtain an OD of 0.3
  • incubation for ca. half an hour shaking at 37°C
  • OD measurement until OD of approximately 0.5 was reached
  • one culture of each colony was induced with IPTG in a final concentration of 1 mM, the other was left uninduced
  • incubation o/n at 30°C, shaking

2015/09/15

GFP measurement with UV light (JN)

  • expressed GFP was visible with the bare eye as well as under UV light
  • one liquid culture was pelleted, 1 ml for following SDS-PAGE, the rest to be prepped
Liquid culture pellet of the same colony. One culture was induced with IPTG, the other not.

SDS-PAGE (Protein Purification Labjournal JD)

  • 1 ml of liquid culture was pelleted and the supernatant discarded
  • 300 µl of 2.5x SDS Loading-dye was added
  • boiling at 95°C for 10min, cooldown
  • 20 µl were loaded on a 12.5% SDS-PAGE gel
  • analysis via coomassie staining
SDS-PAGE of pOP, enrichment of expressed GFP (27 kDa) visible from the induced culture