Team:BGU Israel/Experiments
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Experiments
Read our well-documented notebook.
Week 1 29.07.2013 - 31.07.2013
- DNA volume - 1 uL.
- cuvette width - 0.2 cm.
- Time constant (Tao) - 5.5 msec.
- Two LB agar plates with cmp were plated with bacteria - one with 20 uL of culture, the other with 50uL. The plates were grown overnight in 300c, and on both colonies grew.
- 51 uL DDW.
- 15 uL KAPA HIFI buffer.
- 2.25 uL of dNTPs.
- 2.25 uL of forward primer.
- 2.25 uL of reverse primer.
- 1.5 uL of KAPA polymerase.
- The annealing temperature was set to 450C.
![](https://static.igem.org/mediawiki/2013/3/35/BGU_lab1.jpg)
Week 2 01.08.2013 - 10.08.2013
- 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
![](https://static.igem.org/mediawiki/2013/d/da/BGU_ex42.png)
- Control sample was prepared – containing all components except the template.
- Each reaction was made twice.
![](https://static.igem.org/mediawiki/2013/7/7c/BGU_ex43.png)
![](https://static.igem.org/mediawiki/2013/8/84/BGU_ex44.png)
![](https://static.igem.org/mediawiki/2013/1/1d/BGU_ex45.png)
- 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.
![](https://static.igem.org/mediawiki/2013/3/33/BGU_ex46.png)
- Control sample was prepared – containing all components except the template.
- 10 samples were made – one from each colony.
![](https://static.igem.org/mediawiki/2013/0/0d/BGU_ex47.png)
![](https://static.igem.org/mediawiki/2013/f/fb/BGU_ex48.png)
- 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
![](https://static.igem.org/mediawiki/2013/8/89/BGU_ex49.png)
![](https://static.igem.org/mediawiki/2013/5/51/BGU_ex50.png)
![](https://static.igem.org/mediawiki/2013/4/42/BGU_ex51.png)
![](https://static.igem.org/mediawiki/2013/e/ed/BGU_ex52.png)
- 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 3 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.
![](https://static.igem.org/mediawiki/2013/b/b2/BGU_ex36.png)
![](https://static.igem.org/mediawiki/2013/6/62/BGU_ex37.png)
- 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
![](https://static.igem.org/mediawiki/2013/5/58/BGU_ex38.png)
![](https://static.igem.org/mediawiki/2013/9/9a/BGU_ex39.png)
- 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.
![](https://static.igem.org/mediawiki/2013/d/d2/BGU_ex40.png)
![](https://static.igem.org/mediawiki/2013/a/a3/BGU_ex41.png)
![](https://static.igem.org/mediawiki/2013/7/70/BGU_lab4.jpg)
Week 4 18.08.2013 - 04.08.2013
- BL21 pkD78 + puc57-CI
- BL21+ puc57-Time Bomb
- BL21 + puc57-CI
![](https://static.igem.org/mediawiki/2013/b/bd/BGU_lab5.jpg)
Week 5 25.08.2013 - 31.08.2013
![](https://static.igem.org/mediawiki/2013/6/68/BGU_ex26.png)
![](https://static.igem.org/mediawiki/2013/4/4c/BGU_ex27.png)
![](https://static.igem.org/mediawiki/2013/0/0a/BGU_ex28.png)
![](https://static.igem.org/mediawiki/2013/2/22/BGU_ex29.png)
![](https://static.igem.org/mediawiki/2013/5/52/BGU_ex30.png)
![](https://static.igem.org/mediawiki/2013/7/72/BGU_ex31.png)
![](https://static.igem.org/mediawiki/2013/7/72/BGU_ex32.png)
![](https://static.igem.org/mediawiki/2013/5/5f/BGU_ex33.png)
![](https://static.igem.org/mediawiki/2013/4/44/BGU_ex34.png)
![](https://static.igem.org/mediawiki/2013/6/6a/BGU_ex35.png)
![](https://static.igem.org/mediawiki/2013/2/26/BGU_lab2.jpg)
Week 6 01.09.2013 - 07.09.2013
![](https://static.igem.org/mediawiki/2013/2/24/BGU_ex25.png)
Week 7 08.09.2013 - 14.09.2013
![](https://static.igem.org/mediawiki/2013/2/26/BGU_ex19.png)
![](https://static.igem.org/mediawiki/2013/6/6f/BGU_ex20.png)
![](https://static.igem.org/mediawiki/2013/a/a6/BGU_ex21.png)
![](https://static.igem.org/mediawiki/2013/8/8f/BGU_ex22.png)
![](https://static.igem.org/mediawiki/2013/2/29/BGU_ex23.png)
- crbR 1 - 40 ng/μl
- crbR 2 - 10 ng/μl
- GK cassette - 17.5 ng/μl
![](https://static.igem.org/mediawiki/2013/0/0b/BGU_ex24.png)
- 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 8 15.09.2013 - 21.09.2013
![](https://static.igem.org/mediawiki/2013/6/69/BGU_ex17.png)
![](https://static.igem.org/mediawiki/2013/6/69/BGU_ex18.png)
- 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)
![](https://static.igem.org/mediawiki/2013/3/33/BGU_ex1.png)
![](https://static.igem.org/mediawiki/2013/b/b4/BGU_ex2.png)
![](https://static.igem.org/mediawiki/2013/c/cf/BGU_ex3.png)
![](https://static.igem.org/mediawiki/2013/3/35/BGU_ex4.png)
![](https://static.igem.org/mediawiki/2013/1/1d/BGU_ex5.png)
![](https://static.igem.org/mediawiki/2013/5/58/BGU_ex6.png)
![](https://static.igem.org/mediawiki/2013/8/80/BGU_ex7.png)
![](https://static.igem.org/mediawiki/2013/c/ca/BGU_ex8.png)
![](https://static.igem.org/mediawiki/2013/e/ea/BGU_ex9.png)
![](https://static.igem.org/mediawiki/2013/a/ae/BGU_ex10.png)
![](https://static.igem.org/mediawiki/2013/f/fc/BGU_ex11.png)
![](https://static.igem.org/mediawiki/2013/1/1b/BGU_ex12.png)
![](https://static.igem.org/mediawiki/2013/f/f4/BGU_ex13.png)
![](https://static.igem.org/mediawiki/2013/3/34/BGU_ex14.png)
![](https://static.igem.org/mediawiki/2013/6/6d/BGU_ex15.png)
![](https://static.igem.org/mediawiki/2013/4/41/BGU_ex16.png)
- 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.