Difference between revisions of "Team:Freiburg/Labjournals/Cloning/September"

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

• incubation at 37°C, ~1h

PCR Purification of EcoRI digest (JN)

• Qiagen kit
• eluted in 20µl dH2O

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

• 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)

• 37°C for 1h

PCR Purification of pOP Digest

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

pOP digest with PstI

• 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

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

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

• 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

Miniprep: pOP (LS)

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

Digest: pOP and K592009 with EcoRI (LS)

• 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)

• 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

Mini-Prep of K592009 (JN)

• Qiagen kit
• eluted in 20µl dH2O

Digest of all three preps with EcoRI (JN)

• incubation at 37°C for 1h

PCR clean-up (JN)

• Qiagen kit
• eluted in 20 µl dH2O

Digest: K592009 with PstI (JN)

• 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)

• 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

Clean-up of pOP EcoRI digest (JN)

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

Subsequent digest with PstI (JN)

• 37°C for 1h
• analysis on 1% agarose gel, see below

Digest of pOP_Anderson_GFP with EcoRI (JN)

• 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)

• 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

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

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

Test-digest of pOP_K592009 with EcoRI (JN)

• 37°C for 1h

Clean-up of pOP_K592009 EcoRI digest (JN)

• Qiagen kit
• eluted in 20µl dH2O

Subsequent digest with PstI (JN)

• 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)

• 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)

• 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)

• 1h at RT

Trafo of pOP_I13504 into BL21 (LS)

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

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

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

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