Team:Fatih-Medical/Lab/Diary

Diary

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.

Week 9

Saturday

• J04500, K103006 were digestioned and carried out electrophoresis
• Transformations were made for XyIE,pCons and TEV Cleavage Site.
• Digestion was made for TEV N and TEV C from their previous isolated forms.Then electrophoresis was carried out for these parts.

Sunday

• Ligation of TEV N-C and placI was performed and transformed
• Ligation of K103006 and plac3 was performed and transformed
• Isolations of XylE,pCons and TEV Cleavage Site were performed
• Digestion,ligation and transformation of XylE,pCons and placI were performed.
• Presentation which would take place at Dışkapı on thursday was prepared and studied
• We planned to make a video clip of our team

Week 10

Monday

• Laboratory was organized
• Electrophoresis of OmpA-Pro was carried out but probably there was a contamination so the results weren’t what we expected
• Ligations of GFP and OmpA were performed

Tuesday

• Liquid culture of pCons and XylE were performed
• Digestion and electrophoresis of pLac and OmpA were performed
• The list of emergency items were written

Wednesday

• Transformations of J04500, K103006 and E0040 were performed
• Isolation and digestion were performed from liquid culture of pCons+XylE
• The results were correct. Streak was performed from liquid culture of pCons+XylE

Thursday

• Research day

Friday

• Research day

Saturday

• Research day

Sunday

• Research day

Week 11

Monday

• Research day

Tuesday

• Research day

Wednesday

• Research day

Thursday

• Research day

Friday

• Research day

Saturday

• Research day

Sunday

• Research day

Week 12

Monday

• Application was made to TUBITAK 2209-C for sponsorship activities

Tuesday

• Digestion of pLac+OmpA+500 was performed.Then electrophoresis was carried out for these parts.

Wednesday

• Digestions of pLac, OmpA and 500 were performed. Then electrophoresis was carried out for these parts.
• pLac and OmpA results were correct
• Meeting was carried out and procedures would be discussed again

Thursday

• Digestion procedure was changed
• Digestions of pLac and OmpA were performed. Then electrophoresis was carried out for these parts.The results were correct so gel extraction was performed for these parts.
• Although digestion and electrophoresis of 500 was performed again, the result was same; nothing.

Friday

• Research day

Saturday

• Research day

Sunday

• Research day

Week 13

Monday

• Research day

Tuesday

• Research day

Wednesday

• Research day

Thursday

• Liquid cultures of 500 from different dates was prepared.
• Ligation of 59M (pLac+OmpA) was performed

Friday

• Transformations of Ω, Ω2 and 59M were performed
• 4 digestions (two of them are control group) of Ω2 were performed

Saturday

• Research day

Sunday

• Research day

Week 14

Monday

• Research day

Tuesday

• Chl LB Agar was prepared
• PCR Purifications of q,m,t,lux I,z,62a,k,h,60a,58a and w were performed.
• PCR of 4 and 5 was performed
• OD measurements of 60 sample were performed and their names, dates were tagged.
• Pipette calibration was performed.
• Liquid cultures of 5wzt,50m,5wt were performed
• Isolatin and digestion of 59α were performed
• Gel extractions of r1,500,500-2,500-3,r1, α1, α2, α3,0,zt2,r-2k1,r-3k1,r2 were performed
• Digestions of 9,t,w,m,h,z,k,58a, α,60a,62a,luxI,59 α1,59 α2 were performed
• DJColi and MColi were remained for the name voting for our projects second module
• Student certificate and certificate of deposit from university were taken for visa application
• Laboratory turn of duty was redetermined

Wednesday

• Isolations of 5wzt1,5wzt2,5wzt3 were performed

Ligations of 50m and 5wzt were performed

• Digestions and electrophoresis of 9,5wzt1,5wzt2,5wzt3,h,k,t,z, α were performed. All results except from α and k were correct
• PCR of y,GFP, α,c,r and 4 was performed but there was no results
• The sequencing of 59h2,59h3 was performed
• Application forms of schengen visa were filled out

Thursday

• PCR of GFP,y,c,r, α was performed, just r is incorrect
• PCR of k and second PCR of r were performed but both of them is incorrect
• PCR Purification of y,c, α,GFP was performed
• Digestions of y,c, α,GFP,9 was performed, then electrophoresis was carried out for these parts.
• Ligations of 59 α,59z,5w α,5wz,59m,5luxIt were performed
• 59 α was embarked three different vectors, others ( 59z,5w α,5wz,59m,5luxlt) was embarked after-isolation-digestion vector

Friday

• Isolations of 50m,5wzt,59zt were performed
• PCR Purification of r,k was performed
• Digestions of r,k,50m,5wzt,59zt,m were performed, then electrophoresis was performed for these parts
• A meeting was held for Secret Book of FM
• Team continued to work for our video

Saturday

• Isolations of 5w α,59 α,59m,592,5wz,5luxt were performed but the results are incorrect
• PCR of k,b,0,plac,r was performed then parts’ electrophoresis was carried out
• Ligations from two different N.Free Water were performed for control.

Sunday

• Isolations of con(contamination) 1,con 4-1,con4-2,con4-3,59m-1,59m-2,5wz,59 α1,5luxIt2-1,59 α2,5luxIt3 were performed, then digestions and electrophoresis of these parts were performed. Except from 59 α-1,all results are correct.
• Ligations of ak9h (from gel extraction) and ak9h ( from PCR) were performed
• Transformation of N.Free Water ligation for negative control was performed

Week 15

Monday

• Isolations and digestions of 50m1-2 and 59zt were performed then electrophoresis carried out for these parts but the results were incorrect.
• Liquid cultures of ak9h and J04450 were performed
• Transformations of ak9h and mix were performed for control

Tuesday

• Labratory was cleaned
• Isolation of RFP AK1, RFP AK2, RFP AK3, 9h AK GX1, 9h AK GX2, 9h AK GX3, 9h AK PCR1, 9h AK PCR2, 9h AK PCR3 were performed.Then digestions of these parts were performed.
• PCR of zt,b,q,k,mix,plac,luxI,w,u was performed
• Ligation and transformation of 1wzt,19r,1p α,19y,10m,1w α,1pz,19m,1luxt,1pzt,1wz,1ab were performed

Wednesday

• PCR Purifications of pLac,luxI,w,zt,9,k were performed
• Isolations of 19h PCR(1,2,3), 19h PCR(1,2,3) were performed
• Digestions of 9,k,luxI,plac,zt,w,19h PCR(1,2,3),19h GX(1,2,3) were performed, then electrophoresis of these parts were performed

Thursday

• Isolations of 1wzt,1wz,1pzt,100,1w α,1luxt,19y,19r were performed
• Digestions of 1wzt,1wz,1pzt,100,1w α,1luxt,19y,19r were performed, , then electrophoresis of these parts were performed but results are incorrect

Friday

• Isolations, digestions and electrophoresis of 1p α1,1p α3,1pz11pz2,1py1,1py2,1py3 were performed
• Electrophoresis of z,9,r,m,b,h,plac,t,zt,k carried out, only 9 and plac is right
• Digestion of 9 were performed and 9 ligated with h. Then 9h transformed to amp plate.

Saturday

• Isolations of 19hh PCR1-1, 19h PCR 1-2, 19h PCR 1-3, 19h PCR 2-1, 19h PCR 2-2, 19h PCR 2-3, 19h GX1,19h GX2, 19h GX3 were performed, except for 19h PCR 1-1, all of the parts digestioned and electrophoresis was carried out
• PCR of zt,h,b,t,z,c,k,mix,m was performed
• Electrophoresis of 9,9-1,zt,h,b,t,z,c,k,mix,m was performed

Sunday

• Isolations of 9h(1,2,3,4,5) was performed
• There were no colony in plate of P2-GFP
• Digestion of 9h(1,2,3,4,5) was performed
• PCR of u,c was performed
• PCR of Z,h,k,zt,r,m than PCR purification of these parts was performed
• Gel extraction of b,c was performed

Week 16

Monday

• Isolations, digestions and electrophoresis of 1wz1,1wz2,50m1,50m2,19r1,19r3,pCony1-2 were performed but unfortunately the results were incorrect
• PCR of 9,m,t was performed
• PCR Purifications, digestions and electrophoresis of r,t,9,60a were performed
• Transformations and PCR of coming parts (1,2,6,7,8,9,10,11) were performed
• Transformations of 59x,1w α were performed

Tuesday

• Transformations of 1,2,6,7,8,9,10,11 were performed
• Liquid cultures were taken from 1,2,6,7,8,9,10,11
• PCRs and Gel extractions of 1,2,6,7,8,9,10,11 were performed. In the PCR results there were two bants from samples in order to ensure we made a gel extraction
• Digestions of Psb1A3,Psb1K3,400,700 were performed
• Ligations and transformations 4p α,7p α,5p α,19-2,19h-3 were performed
• Digestions and electrophoresis of 1,2,9,10,11 were performed

Digestions and electrophoresis of PCR Purification of Gel extraction of 1,2 were performed

• The labratory was cleaned

Wednesday

• Gradient PCR for heat optimization and decided to 60 Celcius degree for PCR
• Gel extraction of 1,2,9,10,11 were performed
• Isolations, degistions and electrophoresis of 1,2,6,7,8,9,10,11 were performed
• PCR of 1,2,6,7,8,10,11 were performed at 60 Celcius, all bants are right but unfortunately we had unwanted bants. Then digestions of these parts were performed

Thursday

• Ligations of 9-3+GFP+500, 9-3+GFP+100-23, 9-3+GFP+400-3, 9-1+h+500-1, 9-1+h+100-1, 9-1+h+700-2 were performed then these parts transformated.
• Liquid cultures of 19h-1,19h-2,19h-3, 19r-2, 4p α-1,4p α-2,4p α-3,4p α-4,4p α-5,4p α6,7p α, 7p α-1, 7p α-2, 7p α-3 were performed

Friday

• Meeting day

Saturday

• Ligations of TCTN, яnz, яn,9r,9h,luxIt,walfa,wzt,qalfa,qzt,ab,4rfp,7rfp,5gfp generator,4prfp were performed.
• PCR of 7 and 8 were performed.Then they were purificated and digested.
• Liquid cultures were prepared for gfp generator,rfp with rbs, я
• Sequence of 4palfa and 19h were performed.
• Electrophoresis was performed again for я because it’s last electrophoresis result was wrong
• Electrophoresis was performed to control some nameless samples.

Sunday

• PCR products of gama,e,m and q were purificated and then digested.
• wz,wzt,qzt,luxIt,TcTn,qalfa were isolated and digested.
• Electrophoresis was performed for all of these parts.
• Liquid cultures were prepared for 1ab,4ab,49r,4p rbs+rfp,5яgfp
• Ligations of 4qzt,4qalfa,4wgfp,4яnz,4яn,4qgfp were performed.Then all of these parts and 49h were transformed.

Week 17

Monday

• 1ab,4ab,49r,4p rbs+rfp,5яgfp were isolated and digested.
• Electrophoresis was performed for all these parts.
• PCR purification samples of luxI,Tn,w,r,zt,gfp and 500 from isolation were digested.Then electrophoresis was performed for all of these parts and Tc.
• We were thinking our Amp didn’t work, so we spreaded our compotent cells to Amp plate for controling this.
• We decided to make new compotent cells because ?????
• Liquid cultures were prepared for 5palfa
• PCR of Tc,Tn,e,q,r,0 were performed.
• PCR products of Tn,Tc,e,q were purificated

Tuesday

• Compotent cell was made.
• 5palfa was isolated.
• We cleaned our refrigerator.
• Ligations of qzt,qgfp,wzt,wgfp,9h,9r,ab,LuxIt, яn, яnz with 400 and 500 backbones were performed.
• Ligation of TcTn with 400,500 and 700 backbones were performed .
• All of these ligastions were transformed.

Wednesday

• Research day

Thursday

• Research day

Friday

• We made again compotent cell because our last compotent cell experiment wasn’t good enough
• PCR products of TcTn and яn were made.
• Electrophoresis was performed for TcTn, яn and 4luxIt(from digestion).The results were correct.
• PCR products of TcTn and яn were purificated and digested.(the results were good)PCR was performed for 4luxIt but PCR result of 4luxIt wasnt good.
• İsolations were made for 4qzt,4q-gfp,4wzt,4w-gfp,4ab and then all of these parts were digested.
• Electrophoresis was performed and results of 4ab and 4q-gfp(not clear) were correct.
• PCR was made for 4ab and 4q-gfp.
• We started to sequence TcTn and яn.
• Ligations of qzt, яnz,wzt and w-gfp were made to 400 backbone and they were transformed.
• я, q and linear 400 backbone were digested again.(because of low volume)
• LB agar was prepared with Amp.
• Liquid cultures prepared for 49r-1 and 49r-2.
• Transformation was made for controlling new compotent cells.
• Electrophoresis was performed for k,u,0 and 400 backbone.
• PCR was performed for 4ab and 4q-gfp.(4ab was correct)

Saturday

• PCR was performed for 49r.
• 49r,5 яn,5w-gfp,0 were isolated and then digested.Electrophoresis was performed for these parts.
• Liquid cultures were prepared for 19h,4wzt,4qzt,4 яnz,4wgfp
• TcTn’s sequence was correct.
• Compotent cell was controlled with Amp and Chl
• TcTn’s liquid culture was made Co-Ip and SDS-Page
• We threw unnecessary falcons in our fridge

Sunday

• PCR was performed for 4qzt,49r,4ab,4qgfp,4luxIt
• 0,5wgfp, 5 яn were isolated and digested.
• PCR product of 4ab1,4ab2,4luxIt were purificated and digested.
• We were thinking that 9r was correct so we purificated this PCR product and digested.Electrophoresis was performed for these part and Gel extraction was made for correct band.
• We enumerated our old compotent cells and they were transformed for control

Week 18

Monday

• We made a new ladder stock.
• We made SDS-page again because we had made a little mistake.
• Spe1 and Pst1 decreased because of that we made an order for these enzymes.

Tuesday

• PCR was performed for without antibodies Tc and Tn genes
• 5TcTN яnz ,7TcTn яnz,5TcTnяnz-2,4TcTnяnz,4яn,4wgfp, яnz were isolated and digested.
• TcTn was made Co-Ip and SDS-page for negative control.
• Ligations of 1qzt,19r,1wzt,4wrr,4qrr,1py,1яy,5TcTn,9gama,40m,4TcTnяnz were performed and all of this parts were transformed.
• 400 and tcs were digested.
• Electrophoresis was performed for without antibodies Tc and Tn genes and gel extraction was made for this parts.Then this gel extractions simples were ligated with GFP.The last form of this parts 4 TcGfp and 4 TnGfp were transformed.

Wednesday wiki freeze day

• We made gradient PCR for without antibodies Tc and Tn genes.Electrophoresis and gel extraction was performed for this parts.
• PCR was performed for 49gama and 4TcTn яnz.