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(RFC25) 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(RFC25)
Gel check of PCR product and PCR clean up
Digestion of MLS(RFC25) with EcoRI & SpeI, run gel

23-7-2013
MLS(RFC25) Gel purification (Cont), and gel check
pSB1C3 plasmid extraction
Digestion of pSB1C3 with EcoRI & SpeI

24-7-2013
pSB1C3 Gel purification (Cont.)
MLS(RFC25) & pSB1C3 ligation, and transform to CAM plate

25-7-2013
Colony PCR of MLS.pSB1C3(RFC25)
Inoculation of MLS.pSB1C3(RFC25)

26-7-2013
Plasmid extraction of MLS.pSB1C3(RFC25)
Digestion of ligated MLS-pSB1C3 with EcoRI & SpeI, gel check
Gel check for MLS.pSB1C3(RFC25) colony PCR product
Spread the remaining re-suspend of correct colony on CAM plate

29-7-2013
Repeat digestion of pSB1C3.MLS(RFC25) with EcoRI and SpeI, gel check and Sanger sequence
Spread plate of GFP(RFC25) transformed colonies

30-7-2013
pSB1C3.MLS (RFC25) 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(RFC25) colonies

31-7-2013
pSB1C3.MLS (RFC25) Sanger sequence(Cont.)
GFP (RFC25) inoculation

01-08-2013
GFP(RFC25) plasmid extraction
Prepare the glycerol stock and stored it
Digestion of GFP(RFC25) with EcoRI and SpeI, gel electrophoresis

02-08-2013
GFP (RFC25) plasmid extraction
Digestion of GFP(RFC25) with EcoRI and SpeI, gel electrophoresis
Inoculate BBa_J176171 using Amp LB, and BBa_K404108 using CAM LB

5-8-2013
GFP (RFC25) 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 (RFC25)
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 RFC25 format

7-8-2013
MLS (RFC 10 & RFC25) 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(RFC25) digestion with EcoRI & SpeI

8/8/2013
Gel purification of GFP(RFC25) & 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
MLS(RFC10).pSB1C3 & gpf.polyA plasmid extraction
Digestion of MLS(RFC10).pSB1C3 & gpf.polyA with EcoRI-HF and PstI-HF
K929000 gel check
pSB1C3 digestion with EcoRI & PstI
MLS(RFC25).pSB1C3
Streak plate with transformed MLS(RFC25).pSB1C3 to purify colony

14/08/2013
Inoculation of MLS(RFC25).pSB1C3 & GFP.PolyA

15/08/2013
pSB1C3 gel purification
MLS(RFC10).pSB1C3 Sanger sequencing (did not submit as the colony turn out to be not single)
Plasmid extraction of MLS(RFC25).pSB1C3 & GFP
Digestion check of MLS(RFC25) 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 MLS(RFC25).pSB1C3 & gpf.polya
MLS(RFC25).pSB1C3 PCR check
Streak plate with transformed MLS(RFC10).pSB1C3 to get single colony
Inoculation of pEGFP-N1, pCMV/my/mito.GFP, pCMV/myc/mito
Streak plate with transformed pEGFP-N1

19/08/2013
Ligation of CMV and pSB1C3 (1st try)
Transformation of CMV and pSB1C3 into DH5alpha
Streak pCMV/myc/mito.GFP and pCMV/myc/mito.EGFP to AMP plate
MLS(RFC25).pSB1C3 & GFP.polya digestion with AgeI & ApaLI, and transformation of the correct colony
MLS(RFC25).pSB1C3 Sanger Sequencing

20/8/2013
MLS(RFC25).pSB1C3 Sanger Sequencing(Cont.)
Plasmid extraction of pEGFP-N1, pCMV/myc/mito.GFP and pCMV/myc/mito.EGFP
MLS(RFC25).pSB1C3 digestion with AgeI-HF & PstI-HF
GFP.polya.pSB1C3(1st try) digestion with NgoMIV & PstI-HF
Repeat PCR for CMV(2nd try), digestion with EcoRI-HF & PstI-HF, PCR clean up

21/08/2013
Gel purification of GFP/polyA digested with NgoMIV & PstI-HF : too low concentration
EtOH precipitation of GFP/polyA digested with NgoMIV & PstI-HF
Pcmv.pSB1C3(2nd) ligation, and transformation to DH5alpha
MLS(RFC10) bacteria inoculation

22/08/2013
MLS(RFC10).pSB1C3 plasmid extraction, PCR check with VR VF2
Repeat digestion of GFP.polyA with NgoMIV & PstI-HF (2nd) with NgoMIV & PstI-HF
Pcmv.pSB1C3 (3rd) ligation and transformation
Inoculation of Pcmv.pSB1C3 (2nd)
GFP.polyA with NgoMIV & PstI-HF (1st) gel check

23/08/2013
Sanger Sequencing for MLS(RFC10).pSB1C3 and MLS(RFC25).pSB1C3
Pcmv.pSB1C3 (2nd) plasmid extraction, and digestion check with EcoRI & NdeI
Streak the right colony of Pcmv.pSB1C3 (2nd) into plate
GFP.polyA with NgoMIV & PstI-HF (2nd) gel purification

26/8/2013
Sanger sequencing for Pcmv.pSB1C3 (#1), MLS(RFC10).pSB1C3 (#4.3), and MLS(RFC25).pSB1C3 #1.1)
Gel check for GFP.polyA (NgoMIV & PstI-HF) for 1st & 2nd
GFP.polyA digestion with XbaI & PstI-HF, run gel and gel purification
Pcmv digestion with SpeI & PstI-HF, dephosphorylated
MLS(RFC25).pSB1C3 digestion with AgeI & PstI-HF, dephosphorylated
Repeat Sequencing for MLS(RFC25).pSB1C3

27/8/2013
MLS(RFC25).pSB1C3 Sequencing (Cont.)
Repeat Sequencing for Pcmv.pSB1C3
MLS(RFC10).pSB1C3 Sequencing (Cont.), transformed new plate with correct colony (#4.3)
GFP.polyA(XbaI & PstI-HF) gel Purification(Cont.), and gel check
Gel check for Pcmv (SpeI & PstI-HF) & MLS(RFC25).pSB1C3(AgeI & PstI-HF) 
GFP(RFC25) digestion with EcoRI-HF & PstI-HF, transformed new plate for correct colony (#2)

28/8/2013
Ligation of Pcmv (S&P) and GFP (X&P), transformed
Ligation of MLS (A&P) and GFP (N&P), transformed
Pcmv.pSB1C3 and MLS(RFC25) sequence confirmed
GFP(RFC25) gel purification, EtOH precipitation
GFP(RFC25) nanodrop: concentration is too low, abandone

29/8/2013
Inoculation of MLS.GFP.polyA and CMV.GFP.polyA

30/8/2013
Plasmid extraction of MLS.GFP.polyA & CMV.GFP.polyA
Digestion check of MLS.GFP.polyA with EcoRV-HF & NdeI, and CMV.GFP.polyA with EcoRV-HF & AgeI-HF
Transfection of CMV.GFP.polyA #1

2/9/2013
Repeat Digestion check for MLS.GFP.polyA & CMV.GFP.polyA
Gel check of CMV.GFP.polyA: long gel, delayed since not enough time to run gel
Digestion of MLS.GFP.polyA with XbaI & PstI-HF

3/9/2013
Digestion check of CMV.GFP.polyA with EcoRV-HF & AgeI-HF
Run gel and gel purification of MLS.GFP.polyA (X & P)
Pcmv digestion with SpeI and PstI-HF, dephosphorylated and PCR clean up
BBa_J176171 digestion with SpeI & PstI-HF, dephosphorylation and PCR clean up

4/9/2013
GEl purification for  MLS.GFP.polyA

5/9/2013
Gel check for CMV.GFP.polyA (E&P), CMV.pSB1C3 (E&P), and GFP.polyA.pSB1C3(E&P)
Run gel for MLS.GFP.polyA(XbaI&PstI-HF), CMV(SpeI&PstI-HF), BBa_J176171(SpeI&PstI-HF), and CMV.GFP.polyA (EcoRV-HF, AgeI-HF)

6/9/2013
Digestion of CMV.pSB1C3 (1) & GFP.polyA(4) by EcoRI-HF & PstI-HF
Gel check: decided that cmv is defect, will order new primer

9/9/2013
pSB1C3 digestion with EcoRI-HF & PstI-HF
Inoculation of pEGFP-n1 (from confirmed colony spread plate)

10/9/2013
Gel purification of pSB1C3
Plasmid extraction of pEGFP-N1

12/9/2013
Digestion of MLS.GFP.polyA and GFP.polyA with SpeI & PstI, dephosphorylated
Amplify CMV with PCR using new primer