Team:Hong Kong HKUST/notebook/mod3


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Protein Trafficking Module's Notebook

June 2013

Week 4


Inoculation of pCMV/myc/mito


Plasmid extraction for pCMV/myc/mito
Digestion of pCMV plasmid extraction product with PstI-HF and NotI-HF
Gel electrophoresis of pCMV digested product
Gel check for pCMV digestion product


Gel purification of pCMV gel product
Gel check of pCMV gel purified product
PCR clone MLS from pCMV/myc/mito
Transformation of pEGFP-N1 with SURE


Gel check for the MLS PCR product and PCR Clean Up
Digestion of MLS clean up product with EcoRI & PstI-HF
Inoculation of pEGFP-N1


PCR Clean up of digested MLS
Plasmid Extraction of pEGFP-N1
Digestion of pEGFP-N1 with PstI-HF  & NotI-HF


July 2013

Week 1


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


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


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


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


Week 2


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


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


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


Inoculation of pCMV/myc/mito.EGFP
MLS biobrick Sanger sequencing (Cont.)


Plasmid extraction of pCMV/myc/mito.EGFP
pCMV/myc/mito.EGFP transformation check by digesting with PstI & NotI

Spread plate for confirmed colony


Week 3


pCMV/myc/mito.GFP inoculation


Plasmid Extraction of pCMV/myc/mito.GFP
Nanodrop: concentration is not high enough, will repeat inoculation


Inoculate 4 tubes of pCMV/myc/mito.GFP


pCMV/myc/mito.GFP plasmid extraction
PCR clone MLS (RFC25)


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


Week 4


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


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


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


Colony PCR of MLS.pSB1C3(RFC25)
Inoculation of MLS.pSB1C3(RFC25)


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


Week 5


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


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


pSB1C3.MLS (RFC25) Sanger sequence(Cont.)
GFP (RFC25) inoculation


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


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


August 2013

Week 1


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


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


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


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


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


Week 2



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


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
Streak plate with transformed MLS(RFC25).pSB1C3 to purify colony


Inoculation of MLS(RFC25).pSB1C3 & GFP.PolyA


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


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


Week 3


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


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


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


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

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


Week 4


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


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)


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


Inoculation of MLS.GFP.polyA and CMV.GFP.polyA


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


September 2013

Week 1


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


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


GEl purification for  MLS.GFP.polyA


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)


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


Week 2


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


Gel purification of pSB1C3
Plasmid extraction of pEGFP-N1


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


Week 3
Content 1
Week 4
Content 1
Week 5
Content 1