02-07-2013
Gel Check of pEGFP-N1 plasmid extraction product to confirm presence of DNA
Digestion of pEGFP-N1 (with NotI-HF and PstI-HF) 2nd EGFP product:repeat digestion
Inoculation of pEGFP-N1 3rd EGFP: repeat Inoculation
Get pSB1C3.J04450 plasmid extraction product from Bida
Digestion of pSB1C3 with EcoRI and PstI-HF

03-07-2013
Gel electrophoresis of digested EGFP (2nd)
Plasmid extraction of pEGFP-N1 (3rd)
Digetion of pEGFP-N1 (3rd) using NotI-HF and PstI-HF
Ligation of MLS with pSB1C3
Transformation of ligated pSB1C3-MLS

04-07-2013
Gel purification of pEGFP-N1 (2nd) (cont.)
Gel analysis and gel electrophoresis of pEGFP-N1 (3rd)
Run gel for remaining 3rd EGFP (digested)
Ethanol precipitation of pEGFP-N1 (4th) (using remaining 3rd EGFP plasmid)
Digestion of pEGFP-N1 (4th) and gel electrophoresis
Transformation of pEGFP-N1 into DH10B
Colony PCR of MLS biobrick and gel electrophoresis
Inoculation of MLS colony no.2 & spread the remaining re-suspend colony 2 on CAM plate

05-07-2013
pEGFP-N1 troubleshoot
Digestion by PstI-HF, NotI-HF, PstI-HF +NotI-HF and gel check
Inoculate EGFP from transformed new competent cell
Plasmid extraction of MLS Biobrick
Sanger sequencing of MLS

08-07-2013
Result of MLS Sanger sequencing showed that DNA is not enough, the sequencing failed
Inoculation of MLS.PSB1C3
Plasmid extraction of pEGFP-N1
Digestion of pEGFP-N1 with NotI-HF & PstI-HF
Run gel, and it turned out that NotI-HF did not work

09-07-2013
Plasmid extraction of MLS biobrick
Digestion check on MLS.pSB1C3 product with EcoRI & PstI
Sanger sequencing of MLS biobrick
pEGFP-N1 digestion with PstI & NotI

10-07-2013
pcmv.myc.mito & pEGFP-N1 ligation, and transform into SURE on AMP plate
MLS biobrick Sanger sequencing result showed there is no pellet, repeat Sanger sequencing
Run gel for digestion check of MLS.psb1c3 with EcoRI & PstI

11-07-2013
Inoculation of pcmv.myc.mito.EGFP
MLS biobrick Sanger sequencing (Cont.)

12-07-2013
Plasmid extraction of pcmv/myc/mito.EGFP
pcmv/myc/mito.EGFP transformation check by digesting with PstI & NotI

Spread plate for confirmed colony

15/7/2013
pcmv/myc/mito.GFP inoculation

16/7/2013
Plasmid Extraction of pcmv/myc/mito.GFP
Nanodrop: concentration is not high enough, will repeat inoculation

17-7-2013
Inoculate 4 tubes of pcmv/myc/mito.GFP

18-7-2013
pcmv/myc/mito.GFP plasmid extraction
PCR clone MLS (RFC25)

19-7-2013
Run gel for MLS(25) PCR product, PCR clean up
Digestion of the MLS PCR product using EcoRI and PstI, gel check
Digestion of pSB1C3 with EcoRI & PstI, run gel

22-7-2013
Gel purification of pSB1C3
Gel check of pSB1C3: band is too pale, repeat inoculation
Inoculation of pSB1C3.J04450
Repeat PCR of MLS(25)
Gel check of PCR product and PCR clean up
Digestion of MLS(25) with EcoRI & SpeI, run gel

23-7-2013
MLS(25) Gel purification (Cont), and gel check
pSB1C3 plasmid extraction
Digestion of pSB1C3 with EcoRI & SpeI

24-7-2013
pSB1C3 Gel purification (Cont.)
MLS(25) & pSB1C3 ligation, and transform to CAM plate

25-7-2013
Colony PCR of MLS.pSB1c3(25)
Inoculation of MLS.pSB1c3(25)

26-7-2013
Plasmid extraction of MLS.pSB1c3(25)
Digestion of ligated MLS-pSB1C3 with EcoRI & SpeI, gel check
Gel check for MLS.pSB1c3(25) colony PCR product
Spread the remaining re-suspend of correct colony on CAM plate

29-7-2013
Repeat digestion of pSB1C3.MLS(25) with EcoRI and SpeI, gel check and Sanger sequence
Spread plate of GFP(25) transformed colonies

30-7-2013
pSB1C3.MLS (25) digestion with EcoRI and SpeI
Gel electrophoresis of digested pSB1C3.MLS product
Streak plate for pSB1C3.MLS colony 1,2, and 3
Sanger sequencing of pSB1C3.MLS
Streak plate of EGFP(25) colonies

31-7-2013
pSB1C3.MLS (25) Sanger sequence(Cont.)
GFP (25) inoculation

01-08-2013
GFP(25) plasmid extraction
Prepare the glycerol stock and stored it
Digestion of GFP(25) with EcoRI and SpeI, gel electrophoresis

02-08-2013
GFP (25) plasmid extraction
Digestion of GFP(25) with EcoRI and SpeI, gel electrophoresis
Inoculate BBa_J176171 using Amp LB, and BBa_K404108 using CAM LB

5-8-2013
GFP (25) PCR Check with VR VF2, gel check (over ran)
BBa_J176171 digestion using SpeI and PstI-HF to extract the backbone
PolyA / BBa_K404108 digestion using XbaI and PstI-HF
Inoculation of pSB1C3

6-8-2013
Repeat PCR check of GFP (25)
pSB1C3 plasmid extraction
PolyA / BBa_K404108 gel purification and gel check
BBa_J176171 PCR clean up
BBa_K929000 & BBa_J52034 transformation using CAM plate
PCR amplify MLS in RFC 10 and 25 format

7-8-2013
MLS (RFC 10 & 25) PCR clean up
Nanodrop and gel check for the PCR clean up product
Digestion using EcoRI and PstI
PolyA / BBa_K404108 gel check
pSB1C3 digestion with EcoRI & PstI
GFP(25) digestion with EcoRI & SpeI

8/8/2013
Gel purification of GFP25 & pSB1C3, then nanodrop and gel check
polyA digestion with EcoRI and XbaI, and PCR clean up
Inoculation of BBa_K929000 & BBa_J52034

9/8/2013
BBa_K929000 & BBa_J52034 plasmid extraction: J52034 is not transformed
MLS.pSB1C3 & GFP.PolyA ligation, and transform to SURE on CAM plate

12/8/2013

Digestion check of K929000 with ApaLI & NdeI
MLS.pSB1C3 & GFP.PolyA inoculation
Transformation of J52034

13/08/2013
MLS10.psb1c3 & gpf.polyA plasmid extraction
Digestion of MLS10.psb1c3 & gpf.polyA with EcoRI-HF and PstI-HF
K929000 gel check
pSB1C3 digestion with EcoRI & PstI
MLS25.pSB1C3
Streak plate with transformed MLS25.pSB1C3 to purify colony

14/08/2013
Inoculation of MLS25.pSB1C3 & GFP.PolyA

15/08/2013
pSB1C3 gel purification
MLS10.pSB1C3 Sanger sequencing (did not submit as the colony turn out to be not single)
Plasmid extraction of MLS25.pSB1C3 & GFP
Digestion check of MLS25 with EcoRI & PstI; GFP.polya with ApaLI & AgeI HF

16/08/2013
pCMV(Amplify by PCR from pEGFP-N1) PCR clean up
pSB1C3 dephosphorylation
Run gel of MLS25.psb1c3 & gpf.polya
MLS25.psb1c3 PCR check
Streak plate with transformed MLS10.pSB1C3 to get single colony
Inoculation of pEGFP-N1, pcmv/my/mito.GFP, pcmv/myc/mito
Streak plate with transformed pEGFP-N1