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Contents |
Week 1 - June 1st-7th
Friday
Today we started with a briefing from Mustafa where the main action points of the day were discussed. We had been planning the project of this year.We determined our parts that will be necessary:
- K124014 holin
- K559010 halorhodopsin+terminator(X2)
- K112806 endolysine/T4
- K112808 endolysine/holin/CMV/antiholin
These 4 parts was diluted and tagged. Then we coded these parts:
- C1:J23100 cons-promoter
- C2:CMV promoter
- P1:CL promoter
- H1:Holorodopsin
- P2:IPTG
- L1:endolsin
- L2:holin
- l3:eth+antiholin
We prepared 35 plates and 200 ml LB broth for next experiments.
- Transformation (L1,L2,L3)
- C1,C2 streak
- Coloniese are incubated in liquid culture.(C1)
Saturday
- Coloniese are incubated liquid culture.( 1,5 ml AMP ) for :
C1(x2),C2,N1,L2,L3.
- We made this process 2 times because of high mistake risk for first times
Sunday
- Single colony isolation has made for C1-x,C1-y,C2,L1,L2 and L3.
- C1-x and C1-y are digested with EcoR1 and Pst1.
- We prepared liquid culture for C2,L1,L3 again.Because there was not enough DNA for digestion.
Monday
- Single colony isolation has been made for L1,L2 and L3.
- Digestion has been made with EcoR1 and Pst1 for L1,L3 and C2.
- electrophoresis was performed for C1-x,C2,L1,L2 and L3.
- After electrophoresis,results of L1,L3 was true but the others was wrong.
Tuesday
- Digestion has been made with Xba1 and Pst1 for L1 and L3.
- 34 mg/ml chloramphenicol included 49 tubes was prepared.
- Chloramphenicol included 17 plates was prepared.
- IPTG induceble promoter was coded as P2.
- Transformation has been made for H1,L1P2 and L3P2
- We prepared liquid culture for C1,C2,L2
Wednesday
- Single colony isolation has been made for C1,C2,L2
- We prepared liquid culture for C2 again.Because there was not enough DNA for digestion.
- C1 and L2 are digested.
- electrophoresis was performed for C1 and L2.
- The result of C1 was true but the result of L2 was wrong so a new digestion will been performed and after that electrophoresis will been performed for new L2 and old L2.
- We prepared liquid culture for P2L1,P2L3,C2,H1 and L2.
Thursday
- We made isolation for P2L1,P2L3,H1,C2,L2 and then digestion has been made with EcoR1 and Pst1 for all of these parts.After that electrophoresis was performed for these parts.
- The results of P2L1 and P2L3 might be true , we are not sure about this so we need some expert assistance and we need to sequence these part for being sure. Unfortunately the result of L2 was wrong again.
- We prepared a new liquid culture for L2.
Week 2
Friday
- In order of isoation, digestion and electrophoresis was performed to L2 and C2.By the way electrophoresis was performed 2 times for being sure but both of results were wrong.
- Digestion has been made with Xba1 and Spe1 for H1.
- Ligation was performed for P2 and H1 ,C1 and GFP.
Week 3
Wednesday
- Transformation has been made for C2,L2,P2H1 and C1-GFP (unsuccessful)
- We prepared 200 ml LB broth for next experiments.
Thursday
- Transformation has been made for C2,L2,P2H1 and C1-GFP.
- C2 and L2 (successful)
Friday
- We prepared 200 ml LB broth and 15 plates for next experiments
- Transformation was made for P2H1 and C1-GFP.
- We prepared liqued culture for C2 and L2.
Saturday
- Single colony isolation has been made for C2 and L2.
- C2 and L2 are digested with EcoR1 and Pst1.
WEEK 4
Monday
- electrophoresis was performed for C2 and L2.(Unsuccessful)
- We prepared Tfb1 and Tfb2 for compatent cell.
- We prepared liquid culture for C2 and L2.
Tuesday
- Single colony isolation has been made for C2 and L2. - electrophoresis was performed for C2 and L2.
- C2 and L2 are digested with EcoR1 and Pst1.
WEEK 5
Monday
- We added compotent cells to ampicilin included liquid culture and plate.
- We added compotent cells to cloramphenicoli included plate.
- We incubated cloramphenicol resistance bacteria to ampicilin included plate.
- We added compatent cells to plate without antibiotic.
Tuesday
- We prepared 15 CHL plates.
- We tested compatent cell by RFB transformation.
- We prepared liquid culture for preparetion of compatent cell.
Wednesday
- Transformation was made for C2,L2 and C1-GFP.
Thursday
- Ligation was performed for P2 and GFP.
- We prepared liqued culture for L2 and C2.
- We tested new compatent cell by RFB transformation.
- We prepared kanamycin including plate.
Friday
- Single colony isolation was made for C2 and L2.
- C2 and L2 are digested with EcoR1 and Pst1.
- electrophoresis was performed for C2 and L2.(Unsuccessful)
- Ligation was performed for P2H1 with kanamycin.
- We prepared kanamycin including plate.
- We prepared liqued culture for P2GFP and L2.
- Transformation was made for P2H1.
WEEK 6
Saturday
- Isolation was made for L2 and P2-GFP.After that digestion was made with EcoR1 and Pst1 for these parts.Finally electrophoresis was performed to these parts.The result of P2-GFP was true but L2 was wrong.
- We prepared 5 liquid culture for P2H1.
- Digestion was made with EcoR1 and Spe1 for P2-GFP
Sunday
- Isolation was made 5 coloni for 69h.But microcantifuge is broked.
- We prepared liqued culture for J04450(400)
- Transformation was made for J04450(500-700)
- Our parts were renamed.
WEEK 7
Tuesday
- Transformation was made for J04450(500-700)
- Digestion was made J04450(500-700) EcoR1 and Pst-1.
- We diluted Lac-1.
- Transformation was made for Lac-1.
- electrophoresis was performed for J04450(500-700)
- Ligation was performed for 705-79h.
- Transformation was made for 40h,705 and 79h.
Thursday
- We made isolation from liquid culture of 40h for testing contamination.Then we made digestion with EcoR1 and Pst1. After that electrophoresis was performed .
- Ligation was made for 40h and 70h.After that transformation was made for these parts.
- We prepared 3 liquid cultures for each one of LacI,705 and 79h (totally 9 liquid cultures were prepared )
Friday
- Isolation was made for LacI,705 and 79h.Then digestion was made with EcoR1 and Pst1 for 705 and 79h , with EcoR1 and Spe1 for LacI.After that electrophoresis was performed for all of these parts.But results werent good.
Sunday
- We prepared liqued cultures for Zt and K.
- Isolation was made 0,H and U.
- Digestion was made 0,H and U.
- Electrophoresis was performed for 0,H and U.
- Transformation was made W,P,Z,Alpha,Zt,K.
- We prepared compatent cell and AMP plates
WEEK 8
Monday
- We prepared liqued cultures for W,P,Z,Alpha,Zt and k.
- Transformation was made 40hgx,40h and 70h.
- Digestion was made u1,u2 and u3.(unsuccessful)
- Electrophoresis was performed for u1,u2 and u3.
