Team:BGU Israel/Experiments
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- | < | + | <b>Note: </b>We started the project with a different name for each approach. Eventually we came up with "P.A.S.E", but in our notebook we continued using the old ones for comfort sake: GK (Generation Kill) means P.A.S.E 1, and TB (Time Bomb) means P.A.S.E 2.</br><hr/></br></br> |
<b>2.6.2013 </b></br> | <b>2.6.2013 </b></br> | ||
Pellentesque vit habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat Aenean fermentum, it sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. vit Aenean fermentum, Aenean fermentum, Aenean fermentum, Aenean fermentum, elit eget tincidunt condimentum, sagittis tempus.</br></br> | Pellentesque vit habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat Aenean fermentum, it sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. vit Aenean fermentum, Aenean fermentum, Aenean fermentum, Aenean fermentum, elit eget tincidunt condimentum, sagittis tempus.</br></br> |
Revision as of 18:04, 26 September 2013
Week 1 01.06.2013 - 08.06.2013 ********
2.6.2013 Pellentesque vit habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat Aenean fermentum, it sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. vit Aenean fermentum, Aenean fermentum, Aenean fermentum, Aenean fermentum, elit eget tincidunt condimentum, sagittis tempus. 5.6.2013 Pellentesque vit habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat Aenean fermentum, it sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. vit Aenean fermentum, Aenean fermentum, Aenean fermentum, Aenean fermentum, elit eget tincidunt condimentum, sagittis tempus.
Week 2 07.06.2013 - 15.06.2013 *******
Pellentesque vit habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat Aenean fermentum, it sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. vit Aenean fermentum, Aenean fermentum, Aenean fermentum, Aenean fermentum, elit eget tincidunt condimentum, sagittis tempus.
2.6.2013Pellentesque vit habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat Aenean fermentum, it sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. vit Aenean fermentum, Aenean fermentum, Aenean fermentum, Aenean fermentum, elit eget tincidunt condimentum, sagittis tempus.
5.6.2013Pellentesque vit habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat Aenean fermentum, it sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. vit Aenean fermentum, Aenean fermentum, Aenean fermentum, Aenean fermentum, elit eget tincidunt condimentum, sagittis tempus.
Week 3 01.08.2013 - 10.08.2013 *** (partial)
- Centrifuge 10 min at 4200 g and resuspend pellet in 300 μL ice cold 10% glycerol. (final Vol. 1:100)
- Add up to 100ng of the purified PCR product cassette (1 μL) to 40 μL of competent cells. 3 min on ice.
- Transfer the competent cells into an electroporation cuvette.
- Electroporate with Ec2 setting. #1 cuvette-> 5.8m/sec. #2 cuvette-> 5.9 m/sec.
- Plate 50 μL of culture on LB plates containing the appropriate antibiotic and/or selection agent. A serial of dilution was done, we use 104 – 108 to plate on the agar.
- Cuvette #1 was add to LB agar + IPTG 1mM, incubated at 37oC over night.
- Cuvette #2 was add to LB agar + IPTG 1mM+CMP 50mg/ml, incubate at 37oC over night, to confirm loss of pkd78.
- PkD 78
- pET 15b
- HiFi buffer X5
- DDW
- dNTP's
- ampR FWD & REV primer
- cmpR FWD & REV primer
- kappa HiFi polymerase
- DNA template
- Gel Agarose
- Control sample was prepared – containing all components except the template.
- Each reaction was made twice.
- Colony preparation – pick a colony and resuspend in 5 μl DDW PCR.
- Boil for 5 min at 99oC and immediately chill on ice (PCR program: colony_prep).
- Use the 5 μl as template for PCR.
- Control sample was prepared – containing all components except the template.
- 10 samples were made – one from each colony.
- pkd78 – 151.1 μg ∼ 151.1 μl
- CRB gene – 169.8 μg ∼ 169.8 μl
- pkd78 – 455 μl
- CRB gene – 510 μl
- pkd78 – 151 μl
- CRB gene – 170 μl
- pkd78 backbone – 36 ng/μl
- CRB resistance gene – 19 ng/μl
- The C1 plasmid arrived from hy-labs and was diluted. Final concentration - 50 ng\ul.
- Transformation of puc57-cI to super compotent cells (dh5α) by heat shock.
- Transformation of the pGFPuv into dh5α. This pGFPuv has undergone directed mutagenesis in order to add the stop codon.
- Transformation of C.O (copper oxidase) into BL21 that contain the UAA incorporation tRNA and the acRS (acetyl lysine synthethase). The machinery was on pSUP.
Week 4 11.08.2013 - 17.08.2013
- 2 starters containing: 10 ml (LB), 10ul (CRB 100), 10ul (cmp 50), pkD78 + C1
- Kanamycin cassete has arrived.
- kan cassete PCR from pkD4 plasmid ( T annealing= 52oC).
- pkD78-ampR assembly - digestion of PCR products with XbaI and Xhol (FD)
- products were separated on agarose gel and extracted using gel extracting kit.
- BL21 pkD78 + puc57-C1 +CRB100 (10ul) + CMP50 (10ul)
- BL21 puc57-C1 +CRB100 (10ul)
- PkD 78 PCR product
- CRB PCR product
- HiFi buffer X5
- DDW
- dNTP’s
- ampR FWD & REV primer
- cmpR FWD & REV primer
- kappa HiFi polymerase
- DNA template
- Gel Agarose
- Sample 1 – BL21 pkD78 (EC) -> puc57-CI (1ul)
- Sample 2 – BL21(EC) -> puc57-Time Bomb (1.5ul)
- Sample 3 – BL21 (EC) -> puc57-CI (1ul)
- 2* CRB 100 CMP 50
- 5* CRB 100
- 3* KAN 30
- 35 uL DDW
- 10 uL HIFI buffer
- 1.5 uL rev+fwd primer (cI +his tag)
- dNTP 1.5 uL
- 1 uL DNA template (cI plasmid)
- 1uL KAPA polymerase.
Week 5 18.08.2013 - 04.08.2013
- BL21 pkD78 + puc57-CI
- BL21+ puc57-Time Bomb
- BL21 + puc57-CI
Week 6 25.08.2013 - 31.08.2013
Week 7 01.09.2013 - 07.09.2013
Week 8 08.09.2013 - 14.09.2013
- crbR 1 - 40 ng/μl
- crbR 2 - 10 ng/μl
- GK cassette - 17.5 ng/μl
- DPN1 treatment was performed on the PCR products from the 9\9\13. and electroporation was performed on the products and grown on 2 plates (50ul, 150ul)
- starter has been prepared from single colony - pkD78-amp + CRB100. - checking we have succeeded to replace the antibiotic resistance.
- one colony was taken from the plate made on the 10/9 in order to make a starter.
- 2 starters of BL21, pkD78, amp50, 30oC.
- 2 starters of BL21, pkD78 + puc57-cI, cmp50, amp100, 30oC.
- BL21 + pkD78
- BL21 + pkD78 + puc57-cI
Week 9 15.09.2013 - 21.09.2013
- 8 PCR tubes were prepared: 4 from puc57-cI sample, 4 from TB-cassette sample
- 1 PCR tube was used as blank.
- 10 μL of 5XKAPA HiFi Buffer
- 1.5 μL of dNTP mix
- 6.6 μL (125 ng) of Forward Primer + (125 ng) of Reverse Primer (primer is LAC1/araC , amplification PCR product at 8.9.13)
- 1 μL (5-50 ng) of DNA template (template is pGFPuv from 9.9.13)
- ddH2O to final concentration 50 μL
- Then add 1 μL of KAPA HiFi Polymerase.
- 10μl of competent cells + 2μl of mutagenesis product incubate on ice for 30min.
- Cells were transferred to water bath at 42C for 30sec and immediately back to ice for 2 min.
- 200 μl of SOC medium was added.
- Cells incubated at 37C for 1hr.
- Plated X μl on CRB plates over night at 37C. (50 μl plate X2, 100 μl plate X2)
- 10 μL of 5XKAPA HiFi Buffer
- 1.5 μL of dNTP mix
- 2.5 μL (250 ng) of Reverse+ Forward Primers (primer is LAC1/araC , done by Alex at 16.9.13)
- 1 μL (5-50 ng) of DNA template (template is pGFPuv from 9.9.13)
- 35 μL ddH2O to final concentration 50 μL
- Then add 1 μL of KAPA HiFi Polymerase.
- 1.5 μL Dpn1 was added to mutagenesis product
- Incubation at 37C for 1hr.
- 10μl of competent cells + 2μl of restriction product incubate on ice for 30min.
- Cells were transferred to water bath at 42C for 30sec and immediately back to ice for 2 min.
- 200 μl of SOC medium was added.
- Cells incubated at 37C for 1hr.
- Plated Xμl on CRB plates over night at 37C. (50μl plate X2, 100μl plate X2)
- digestion with restriction enzymes PstI and XbaI.
- Running restriction product in 2% agarose gel for B.bone extraction.
- QI Aquik gel extraction kit for purifying DNA from gel. (nanodrop: con. 16ng/ul)
- Ligation of the purifying B.Bone product with: cI T4, TB cassette, His tag+stop codon, KanR and, AmpR. (incubation for 1hr, 33C. stop reaction in 80C)
- Transformation by electroforetion to BL-21, incubation over night.