Contents
Week 1
Day 1- 07/11/16
Made 5mL of 1000x ampicillin stock(50mg/mL) solution, plates
Made LB
Day 2- 07/12/16
Made competent cells
Made 5mL 1000x chloramphenicol stock(100mg/mL), plates
Transformation of bacteria with plasmid from kitplate
Pconst | Bra_J61002 |
RBS | pSB1C3 |
dTerm | pSB3K3 |
GFP-LVA | pSB1C3 |
RBS-GFP-dTerm | pSB1C3 |
Day 3- 07/13/16
Transformations made on the previous day had failed.
Remade chloramphenicol plates after realising that the concentration was wrong. The stock solution made was 3000x not 1000x.
Redid the transformations attempted on the previous day.
Overnight culture of cells for competent cells batch 2
Day 4- 07/14/16
Made competent cells.
Inoculation of cells transformed on the previous day
Day 5- 07/15/16
Miniprep of inoculated cells.
Made glycerol stock of inoculated cells.
Digestion
Pconst | S+P |
dTerm | E+X |
GFP-LVA | E+S |
RBS-GFP-dTerm | X+P |
Week 2
Day 1- 07/18/16
Holiday
Day 2- 07/19/16
Gel purification of gel electrophoresis product (from 07/15/16).
Did a nanodrop of the purified product. The concentration was quite low so the digestion was done again, the bands could not be seen clearly but the gel was cut where the bands were supposed to be there. Some concentration of DNA could be measured after purification.
Ligation Pconst + RBS-GFP-dTerm, GFP-LVA+dterm
Transformation of ligated product and pSB3K3-I20260 (from kitplate 4 18A)
Day 3- 07/20/16
Transformation done on the previous day on the CAM plate had all failed.
AMP plate was covered in cell colonies. It seemed like it was contaminated. Transformation of pSB3K3-I20260 was successful.
Ligation was re-attempted using the digested products from yesterday.
Transformation was done again, this time using competent cells batch 2.
Inoculation of pSB3K3-I20260 and pconst
Day 4- 07/21/16
Transformation of Pconst + RBS-GFP-dTerm had failed (no colonies on plate), GFP-LVA+dterm(cu with seemed to be successful.
Digestion of Pconst + RBS-GFP-dTerm was reattemped.
Inoculation of gfplva-dterm
Day 5- 07/22/16
Gel electrophoresis of product digested on the previous day. For RBS-GFP-dterm, only one band was seen, probably the wrong enzyme was used or the enzyme is damaged.
Made glycerol stock of gfplva-dterm.
Ligated and transformed pconst-RBS-GFP-dterm
Week 3
Day 1- 07/25/16
No colonies for pconst-RBS-GFP-dterm
Digestion of rbs and GFPlva
Made kanamycin stock 5mg/mL
Colony pcr of gfp-lva dterm-> unsuccessful it appeared to be gfp-lva
Day 2- 07/26/16
Ligation of rbs- GFPlva and pconst
Made more CAM plates
Inoculation of pconst-rbs-gfp-dterm, pSB3K3-I20260
Inoculation of cell culture D2, D3, E17, E18 from 2014
Day 3- 07/27/16
PCR of D2, D3, E17, E18-> E17 showed unexpected bands, others were as expected
Miniprep of D2, D3, E17, E18, pSB3K3-I20260
Made new glycerol stock for cell cultures with the above constructs and one for I20260-pSB3K3
Digestion of D2, D3, pconst.
Ligation of pconst-D2, pcont-D3
Transformation of yesterday’s ligation (RBS-GFPlva) and pconst weak(from kit plate4 4-E)
Inoculation of pconst, E17, E18, E6, dterm, RBS
Found out that the LB was contaminated, transformations and inoculations done previously may have been affected.
Day 4- 07/28/16
Transformation of pconst-D2, pconst-D3 were successful
Miniprep of pconst, E17, E18, dterm, RBS . Nothing had grown in the overnight culture of E6.
Made glycerol stock of E17, E18 again as previous one might have been made in contaminated LB
Digestion of GFPlva-pSB1C3 and Pconst-rbs-gfp-dterm (X+P)
Ligation of the above digested parts.
Proper bands did not appear in the gel electrophoresis image of Pconst rbs gfp dterm (it was not cut at all)
It was found out later that the sequencing results of the cut sites of the plasmids were not correct
Transformation of the ligated product
Inoculation of Pconst-D2, Pconst-D3
Week 4
Day 1- 08/01/16
Last week’s transformations were all successful.
Colony PCR rbs-GFPLVA,PconstWeak,E17,E18-> not quite sure for pconst weak, rbs-GFPlva. E17, E18 seem to be ok
Digestion of J23101-D2, J23101-D3, GFPlva-SB1C3 (E+S)
Ligation pSB1C3+PconstD2,pSB1C3+PconstD3
Transformation of the ligated product
Inoculation of RFPlva, PconstWeak, RBS-GFPlva, Pconst-RBS-GFP-dterm,A9,A10
Day 2- 08/02/16
Yesterday’s transformation had all failed
Miniprep (A9,RBS-GFPPlva,RFPlva,Pconst-RBS-GFP-dterm,PconstWeak)
Made glycerol stock
Digestion (rbs-GFPlva, pconst, pconst weak, d2, d3)
PCR(GFPlva,RBS-GFPlva,RBS-GFPlva2,pconstweak)-> Pconstweak seems ok, no bands for GFPlva, incorrect band for gfplva
Messed up gel extraction
Inoculation of A10(amp),PRGD(KAN),D2,D3,pconstweak(CAN)
Day 3- 08/03/16
Miniprep (A10(amp),PRGD(KAN),D2,D3,pconstweak(CAN)
Digestion(Pconst-BBa_J61002,Pconstweak,D2,D3,pconst-D2,Pconst-D3,dterm,GFPlva,RBS-GFPlva,PRGD-pSB3K3). Concentration of the purified product was low, possibly because he wrong buffer was used.
Ligation(PconstD2+3K3,PconstD3+3K3,GFPlva+61002,RBS-GFPlva+dterm,Pconstweak+D2,Pconstweak+D3)
Transformation of ligated product
Inoculation (Pconstweak,PRGD)
Day 4- 08/04/16
Only transformation for RBS-GFPlva-dterm and GFPlva-J61002 was successful
Miniprep(PRGd-3K3,Pconstweak)
PCR (GFPlva-BBa_J61002×2,GFPlva×2,RBS-GFPlva-dterm×2). Rbs-GFPlva-dterm turned out to be just dterm. For other colonies, no bands were shown, extention of the extension time maybe needed.
Digestion (Pconstweak,D2,D3,PRGd-3K3)S+P
The results for the gel electrophoresis of the digested product was not quite right so igation was not done.
Inoculation (PconstWeak,RBS-GFPlva-dterm×3,PconstD2,PconstD3,GFPlva-BBa_J61002×2)
Day 5- 08/05/16
Miniprep (PconstWeak,RBS-GFPlva-dterm×3,PconstD2,PconstD3,GFPlva-BBa_J61002×2).
Digestion (PRGd-3K3,PconstD2,PconstD3,PconstWeak,D2,D3,dterm,GFPlva-61002)
Ligation (PconstWeak-D2,PconstWeak-D3)
Transformation of ligated product
Week 5
Day 1- 08/08/16
PCR of PconstWeak-D2,PconstWeak-D3. Bands for Pconst weak appeared for some of the colonies, while nothing appeared for most others.
Digestion ( PRGd-3K3, pconstD, GFPlva-J61002, rbs)
Made KAN + CAM plates
Gel electrophorsis of digested product. 3k3 was not cut at all, GFPlva was not cut properly, cant see PconstD2. Ecor1 Spe1may be damaged
Transformation RBS-GFPlva
Inoculation PRGd10ml,GFPlva-61002-4,PconstweakD2,PconstweakD3,E17,E18
Preculture for competent cells
Day 2- 08/09/16
Glycerol stock (GFPlva-61002-4,PconstweakD2,PconstweakD3)
Miniprep(PRGd,GFPlva-61002-4,PconstweakD2,PconstweakD3,E17,E18)
Digestion(E17,E18,PconstweakD2,PconstweakD3,PconstD2×2,PconstD3×2,3K3)(E17,E18:vector). E18 is alright, E17 is too light, no correct bands for Pconst D2 PCR(RBS-GFPlva×3,PconstWeakD2×2,PconstWeakD3×2). All rbsgfplva turned out to be rbs. PconstweakD2,PconstweakD3 seems to be correct.
Made competent cells
Ligation and transformation(PconstweakD2E17, PconstweakD3E18, PconstD2E17, PconstD3E18, PconstD2E18,PconstD3E17,E17-3K3,E18-3K3,CFPlva(kitplate4-5D))
Attempted double transformation(PRGd-3K3,E17-pSB1C3)
Day 3- 08/10/16
Miniprep(RBS-GFPlva×3)
Day 4- 08/11/16
Public Holiday
Day 5- 08/12/16
Public Holiday
Week 6
Day 1- 08/15/16
Public Holiday
Day 2- 08/16/16
Public Holiday
Day 3- 08/17/16
PCR (PconstweakD2E17, PconstweakD3E18, PconstD2E17, PconstD3E18, PconstD2E18,PconstD3E17,E17-3K3,E18-3K3). -> most did not seem to contain the right construct. Except rbs-gfplva. Perhaps the Spe1 has gone bad.
Made CAM plates
Check of restriction enzymes. All the enzymes seemed to work fine.
Inoculation (E18&PRGD(CAM)(KAN)(CAM+KAN), CFPlva(AMP), RBS-GFP-dterm(CAM))
Day 4- 08/18/16
All cultures grew for the inoculation. Double transformation seemed be successful.
Miniprep (E18+PRGd,CFPlva)
Digestion (RBS-GFPlva, RBS-GFP-dterm, dterm, E18+PRGd, CFPlva, Pconst, Pnrd). All except Pconst had undigested fragments. The one with double transformation also showed proper bands
PCR (PconstD2-3K3, PRGd-3K3.) Showed mysterious band of slightly less than 500bp.
Ligation(RBS-GFPlva-dterm, RBS-GFPlva-pSB1A2, Pnrd-61002, Pconst-RBS-GFP-dterm, Pnrd-1A2, RBS-GFPlva)
Transformation (RBS-GFPlva-dterm, RBS-GFPlva-pSB1A2, Pnrd-61002, Pconst-RBS-GFP-dterm, Pnrd-1A2, RBS-GFPlva)
Inoculation (dterm, Pconst, PconstD2, RBS)
Day 5- 08/19/16
PCR (PconstD2-3k3,PRGd-3K3(plasmid),Pconst-RBS-GFP-dterm-1C3, Pconst-RBS-GFPlva). Pconst-RBS-GFPlva seems ok. PconstD2-3k3 is possibly PRGd-3k3. Pconst-RBS-GFP-dterm is ok)
Miniprep (Pconst,pconstD2,RBS,dterm)
Digestion(E17,E18,PconstD2,PconstD3,RBS-GFPlva, dterm, Pconst-61002,PRGd-3K3,CFPlva-1A2). Digestion results are still not too good, some plasmid remains uncut.
Transformation E17PconstD2, E18PconstD2, E17PconstD3, E18PconstD3, Pnrd-1C3, RBSGFPlvadterm(CAM), PconstD2-3K3, PconstD3-3K3(KAN), Pnrd-61002, RBSGFPlva-1A2(AMP)
Week 7
Day 1- 08/22/16
PCR (E17PconstD2, E18PconstD2, E17PconstD3, E18PconstD3, Pnrd-1C3, RBSGFPlvadterm(CAM), PconstD2-3K3, PconstD3-3K3(KAN), Pnrd-61002, RBSGFPlva-1A2(AMP))
Made 3% agarose gel
Made AMP plates
Transformation(PconstD2-3K3, E17-PconstD2, E17PconstD3, RBSGFPlvadterm)
Preculture(160818:Pconst-RBS-GFP-dterm(AMP), RBS-GFPlva-dterm(CAM) 160819:E18-PconstD2(光る),Pnrd1C3×2, E17-PconstD3, E18-PconstD2(CAM), PconstD3-3K3(光らない)(KAN), Pnrd-61002, RBS-GFPlva-dterm(AMP))
Preculture for competent cells
Day 2- 08/23/16
Only PconstD2-3K3, RBS-GFPlva-dterm grew colonies for yesterday’s transformation.
MIniprep (160818:Pconst-RBS-GFP-dterm(AMP), RBS-GFPlva-dterm(CAM) 160819:E18-PconstD2(光る),Pnrd1C3×2, E17-PconstD3, E18-PconstD2(CAM), PconstD3-3K3(KAN), Pnrd-61002, RBS-GFPlva-dterm(AMP))
Digestion(E17.E18,PconstD2,PconstD3,PRGd-3K3). Pconst D2 was not cut. No bands apeared for ToeholdGFPlva and PconstTrigger Made competent cells
Ligation E17+PconstD3, 1A2+Pnrd, RBS-GFPlva+dterm
Transformation (E17PconstD3, RBS-GFPlva-dterm(CAM), Pnrd-1A2, PconstTrigger1, ToeholdGFPlva(AMP))
Double transformation(PcosntD3-3K3+E18(KAN+CAM))
Preculture(PconstD2, PRGd-3K3, E17, E18)
Day 3- 08/23/16
Miniprep(PconstD2, E17, E18, PRGd-3K3)
PCR(PconstD2-3K3(not OK; unknown band seen at around 400bp), Pnrd-1A2(ok), RBS-GFPlva-dterm(ok), E18+PconstD3(not ok))
Digestion(PcosntD2, PcosntD3, PconstWealD2, PcosntWeakD3, E17, E18, PRGd3K3)
Ligation(PconstD2-E17, PconstD2-E18, PconstD3-E17, RBSGFPlva-dterm)
E17-PconstD maybe slightly containated with E18.
Transformation(E17-PconstD2, E18-PconstD2, E17-PconstD3, RBSGFPlva-dterm)(CAM)
Preculture(PconstTrigger1, toeholdGFPlva, Pnrd-1A2(AMP), PconstD2-3K3(KAN), PconstD3-3K3+E18(CAM+KAN)PcWD2, PcWD3(CAM))
Day 4- 08/24/16
Glycerol stock (PconstTrigger1, toeholdGFPlva, Pnrd-1A2, PconstD2-3K3 PconstD3-3K3+E18)
Miniprep(PconstTrigger1, toeholdGFPlva, Pnrd-1A2, PcWD2, PcWD3, PconstD2-3K3)
PCR(E17-PconstD3(not ok), PconstD2-3k3(not ok), PconstD3-3K3, PRGd-3K3, RBS-GFPlva-dterm(not ok))
Digestion(toeholdGFPlva,Pnrd1A2, RBSGFPdterm)(E17,E18,PconstD2,PconstD3,PcWD2,PcWD3)
Ligation(toeholdGFPlva-dterm, RBS-GFPlva-dterm, Pnrd-1C3, E17PcWD2, E18PcWD3, PcWD2-3K3, PcWD3-3K3, PconstD2-3K3)
Made AMP plates
Transformation(toeholdGFPlva-dterm, RBS-GFPlva-dterm, Pnrd-1C3, E17PcWD2, E18PcWD3(CAM), PcWD2-3K3, PcWD3-3K3, PconstD2-3K3(KAN), PconstD23K3+E17(CAM+KAN))
Preculture(E17PconstD3×2 )
Day 5- 08/25/16
Glycerol Stock (E17PconstD3-6)
Miniprep(E17PconstD3-6,-7)
PCR(PcWD3-3K3(unknown), PcWD2-3k3(unknown), E18PcWD3(not ok), E17PcWD2(not ok), E17PconstD2(not ok), E17PconstD3(not ok))
Digestion(dterm-3K3, PconstRBSGFPdterm-1A2)
Ligation(RBSGFPlva-dterm, toeholdGFPlva-dterm, Pnrd-1A2, PconstRBSGFPdterm-1C3)
Transformation(RBSGFPlva-dterm, toeholdGFPlva-dterm, PconstRBSGFPdterm-1C3(CAM), Pnrd-1A2(AMP), PconstD2-3K3+E17(KAN+CAM))
Week 8
Day 1- 08/29/16
PCR(RBS-GFPlva-dterm(ok), toeholdRBSGFPlva-dterm(not ok), Pnrd-1A2(not ok), PconstWD3-3K3(not ok), PconstWD2-3K3(not ok), E18-PconstD2(not ok))
Digestion(PconstRBSGFPdterm1A2, Pnrd, sigma16, sigma35, antisigma16, antisigma33, dterm, E18-1C3, RBSGFPlva-1A2)
Ligation (Pnrd-1C3)
Transformation(Pnrd-1C3, dterm)
Preculture(D8, D9) (E18PcWD3, E17PcWD2, PcWD2-3K3, PcWD3-3K3, RBSGFPlva-dterm)
Day 2- 08/30/16
Glycerol Stock (E18PcWD3, E17PcWD2, RBSGFPlva-dterm, D8, D9)
Miniprep(E18PcWD3, E17PcWD2, RBSGFPlva-dterm, D8, D9)
PCR(PcWD3-3K3, PcWD23K3, toeholdGFPlvadterm(NG), Pnrd1C3(ok), PRGd-3K3(ok))
Digestion(PconstRBSGFPdterm, RBSGFPlva-dterm, D8, D9, Pconst)
Ligation(Pconst-D8, Pconst-D9, Pconst-RBS-GFPlva-dterm, sigma16-1A2, sigma35-1A2, antisigma16-1A2, antisigma33-1A2, Pnrd-1A2, PconstRBSGFPlva-1C3)
Transformation (Pconst-D8, Pconst-D9, Pconst-RBS-GFPlva-dterm, sigma16-1A2, sigma35-1A2, antisigma16-1A2, antisigma33-1A2, Pnrd-1C3, PconstRBSGFPlva-1C3)
Preculture (Pnrd-1C3, PconstRBSGFPdterm, PconstD3, PcWD2-3K3, PcWD3-3K3、PcD2-3K3)
Day 3- 08/31/16
Glycerol Stock (Pnrd-1C3, PcD2-3K3)
PCR(Pconst-D8, Pconst-D9, Pconst-RBS-GFPlva-dterm, sigma16-1A2, sigma35-1A2, antisigma16-1A2, antisigma33-1A2, Pnrd-1C3(ok), PconstRBSGFPdterm-1C3, PconstD2-3K3 )
Miniprep(Pnrd-1C3, PconstRBSGFPdterm, PconstD3, PcD2-3K3)
Digestion(PconstRBSGFPdterm, Pnrd-1C3, RBS, PRGd-3K3, RBSGFPlvadterm, RBSGFPdterm, PcWD2-3K3, PcWD3-3k3)
Transformation (PcWD2-3k3, PcWD3-3k3, sigma16-1A2, sigma35-1A2, antisigma16-1A2, antisigma33-1A2)
Preculture(PconstD8, PconstD9, PconstRBSGFPlvadterm, sigma35-1A2, Pnrd-1C3, PconstRBSGFPdterm-1C3)
Double transformation(E17-PcD2)
Day 4- 09/01/16
LB was contaminated. Made new batch of LB.
Double transformation failed
PCR(Pnrd-1C3(ok), PconstRBSGFPdterm1C3, sigma35(NG))
Digestion(PconstRBSGFPdterm1A2, sigma16, sigma35, antisigma16, antisigma33, Psigma16, Psigma30, E17, PconstD2)
Transformation(sigma16-1A2, sigma35-1A2, antisigma16-1A2, antisigma33-1A2, Psigma16-1A2, Psigma30-1A2, E17PconstD2)
Preculture(PconstRBSGFPlvadterm, PconstD8, E17, JM109, PcWD2)
Day 5- 09/02/16
Glycerol Stock (Pnrd-1C3, PconstRBSGFPlvadterm)
PCR (sigma 16(ok), antisigma33(ok), Psigma16(unknown), PcWD2-3k3(unknown))
Digestion(Pnrd-1C3, PconstD8, PconstD9, PconstD2, E17, E18, sigma35-1A2(0901)
Ligation(Pnrd-RBSGFPdterm, Pnrd-RBSGFPlvadterm, PconstD8E17, PconstD9E18, PconstD2E17, Psigma30-1A2, sigma35-1A2)
Transformation(Pnrd-RBSGFPdterm, Pnrd-RBSGFPlvadterm, PconstD8E17, PconstD9E18, PconstD2E17, Psigma30-1A2, sigma35-1A2)
Week 9
Day 1- 09/05/16
PCR sigma35-1A2(NG), E18PconstD9(OK), E17PconstD8(OK)
Digestion(Pnrd-1A2, PconstD2, D2, Psigma30, Pconst, PcW)
Transformation(Pconst+D2, Pnrd+RBSGFPlvadterm, PcW+RBSGFPdterm, PcW+RBSGFPlvadterm, Psigma30+1A2)
Preculture (PcWD2-3K3, PcWD3-3K3, sigma16-1A2, sigma35-1A2, Psigma16-1A2, antisigma16-1A2, antisigma33-1A2, E17PconstD8, E18PconstD9, JM109)
Day 2- 09/06/16
Glycerol Stock (sigma16-1A2, sigma35-1A2, Psigma16-1A2, antisigma16-1A2, antisigma33-1A2, E17PconstD8, E18PconstD9)
PCR(E178(ok), E17PconstD8(ok), E18(ok), E18PconstD9(ok))
Miniprep(sigma16-1A2, sigma35-1A2, Psigma16-1A2, antisigma16-1A2, antisigma33-1A2, E17PconstD8, E18PconstD9)
Digestion(Pnrd, RBSGFPdterm, RBSGFPlvadterm, Pconsttrigger1dterm, toeholdGFPlva, PconstRBSGFPdterm, dterm, sigma16, sigma35, antisigma16, antisigma33, Psigma16, RBS)
ligation(PcW-RBSGFPdterm, Pconsttrigger1dterm-1C3, toeholdGFPlva-dterm, RBS-sigma16, RBS-anti16, RBS-anti33)
Transformation(PcW-RBSGFPdterm, Pconsttrigger1dterm-1C3, toeholdGFPlva-dterm, RBS-sigma16, RBS-anti16, RBS-anti33)
Day 3- 09/07/16
Holiday
Day 4- 09/08/16
PCR (PcW-RBSGFPdterm(ok), Pconsttrigger1dterm-1C3(NG), toeholdGFPlva-dterm(NG), RBS-sigma16(NG), RBS-anti16, RBS-anti33(ok))
Digestion(sigma351A2, sigma35,Psigma30, Pnrd, RBSGFPlvadterm, RBSGFPdterm, PconstRBSGFPdterm、RBS)
Ligation (pnrd-rbsgfpdterm, Pnrd-rbsgfplvadterm, rbs sigma 35, psigma30-1A2)
Transformation of ligated product
Preculture(PconstD2, PcW-RBSGFPdterm, Pconsttrigger1dterm-1C3, toeholdGFPlva-dterm, RBS-sigma16, RBS-anti16, RBS-anti33)
Day 5- 09/09/16
Glycerol stock (PconstD2, PcW-RBSGFPdterm, Pconsttrigger1dterm-1C3
Miniprep (PconstD2, PcW-RBSGFPdterm, Pconsttrigger1dterm-1C3)
Digestion (pconst, rbs anti16, rbs anti33, rbs sigma16, toeholdgfplva)
Ligation (Pconstrbs-anti33)
Transformation PconstRBS-anti33
Protocol
Miniprep
Miniprep is done using the Wizard® Plus SV Minipreps DNA Purification System from Promega.
Add 1.5mL of overnight culture in a microcentrifuge tube
Centrifuge at 15000 rmp for 1min
Discard suspension
Add 1mL of overnight culture
Centrifuge at 15000 rmp for 1min
Discard suspension
Add 250uL of Cell Resuspension Solution
Vortex
Add 250uL of Cell Lysis Solution
Invert
Add 10uL of Alkaline Protease
Invert
Leave for 3min
Add 350uL of Neutralization solution
Centrifuge at 15000rmp for 10min
Place column in collection tube and transfer all suspension to the column
Centrifuge at 15000rmp for 1min
Discard flow-through
Add 750uL of Wash Solution
Centrifuge at 15000rmp for 1min
Add 250uL of Wash Solution
Set the column in a new microcentrifuge tube and discard the collection tube
Add 100uL of Nuclease-free water to the column
Leave for 1min
Centrifuge at 13000rmp for 1min
Digestion
Add the following in a 1.5mL microcentrifuge tube (from small to larger volume) and incubate at 37℃ for 2 hours.
DNA | 500ng |
10x Buffer | 2uL |
Restriction Enzyme | 0.5uL each |
MilliQ | up to 20uL |
Gel Electrophoresis
1. Prepare agarose gel
1.1 Measure appropriate amount of agarose (1-2%) to 200mL of TAE in a beaker
1.2 Microwave until all agarose has dissolved
1.3 Allow agarose solution to cool down
1.4 Add 10uL of ethidium bromide
1.5 Pour solution into gel tray.
1.6 Leave until gel has solidified
2. Add corresponding amount of 6x loading dye (4uL to 20uL of digestion mixture) to each sample.
3. Prepare ladder
Promega 1kb/100bp DNA Ladder 5uL
Loading Dye 1uL
4. Place agarose gel in electrophoresis machine and fill with 1x TAE until the gel is covered
5. Load samples in the well
6. Run gel at 110V for 30min-1hour
Gel Purification
Cut out desired band visualised under UV
Place in 1.5mL microcentrifuge tube
Add 400uL of Membrane Binding Solution
Incubate at 60℃ for 10min. Vortex every 2 min to melt gel.
Set column in collection tube and apply solution into the column
Leave for 1min
Centrifuge at 12000rpm for 1min
Discard flow-through and add 750uL of Membrane Wash Solution
Centrifuge at 12000rpm for 1min
Discard flow-through and add 500uL of Membrane Wash Solution
Centrifuge at 12000rpm for 5min
Set column in new microcentrifuge tube
Add 20uL nuclease free water
Leave for 1min
Centrifuge 15000rpm for 1min
Ligation
Add the following in a microcentrifuge tube and incubate at room temperature for 15min (for ° end ligation ), 5min (for cohesive end ligation).
Vector | 100-200ng |
Insert | 2-3 times molarity of vector |
T4 DNA Ligase | 1uL |
2x Rapid Ligation Buffer | 5uL |
MilliQ | up to 10uL |
Inoculation
The 1st ,2nd,and 3rd procedures are conducted in clean bench.
Add 4mL LB in a test tube.
Add 4uL AMP(50ug/mL),1.3uL KAN(35ug/mL),or CAM(33mg/mL) into the LB.
Pick colonies or glycerol stock and pipette in the LB.
Incubate at 37℃ overnight.
Heat-Shock Transformation
Add 10uL of DNA/ligation product into 100uL of competent cell
Leave on ice for 30min
Heatshock at 42℃ for 45 seconds
Leave on ice for 2min
Add 1mL of LB (skip this step for AMP)
Incubate at 37℃ for 30 min
Centrifuge for at 15000rpm for 1min
Remove 800uL of suspention
Spread on plate
Incubate at 37℃ overnight