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Contents
August
August 3, 2010
(in Lab: HB, AV, JV)
Objective: To restrict pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet
Method: Used Restriction of Plasmid DNA protocol.
- A front verctor was made made in sRBS ,mRBS, dT plasmids using EcoRI and Xbal enzymes
- pDNA that was cut out of plasmid for a front vector was pBAD, TetR, dT and pLacI using EcoRI and SpeI enzymes
- A back vector was made in sRBS and mRBS plasmids with PstI and SpeI
- pDNA that was cut out of plasmid for a back vector was TeTR and it was restricted with XbaI and PstI
Construct pDNA buffer Enzymes pBAD-sRBS/mRBS pBAD Red EcoRI and SpeI pBAD-sRBS/mRBS sRBS Orange XbaI and EcoRI pBAD-sRBS/mRBS mRBS Orange XbaI and EcoRII sRBS/mRBS-TetR sRBS Red PstI and SpeI sRBS/mRBS-TetR mRBS Red PstI and SpeI sRBS/mRBS-TetR TetR Tango XbaI and PstI TetR-dT TetR Red EcoRI and SpeI TetR-dT dT Orange XbaI and EcoRI dT-pTet dT Red EcoRI and SpeI dT-pTet pTet Orange XbaI and EcoRI pLAcI-sRBS/mRBS pLacI Red EcoRI and SpeI pLAcI-sRBS/mRBS sRBS Orange XbaI and EcoRI pLAcI-sRBS/mRBS mRBS Orange XbaI and EcoRI Mms6-PET28(a) PET28(a) Orange NotI
- For all reactions
- 158 (µL) Milli-Q H2O
- 10 (µL) Buffer
- 0.5(µL) of each enzyme
- 10 (µL) pDNA
Restriction was incubated for 1 hour at 37oC
Objective: Run PCR of pBAD, TetR, dT, pLacI, and mms6.
Method: Used PCR thermocycler iGEM program 7
Component Volume per tube (µL) Master Mix (x6) MilliQ H2O 41.8 250.8 10x Pfu Buffer with MgSO4 5 30 dNTPs 1 6 Forward Primer 0.5 3 Reverse Primer 0.5 3 Template DNA 1 - Pfu DNA polymerase 0.2 - Total 50 292.8
- Added 48.8 µL of Master Mix to each PCR reaction
Objective: Complete maxipreps of Lumazine (K249002), EYFP (E0030), and ECFP (E0020) and run on 1% agarose gel.
Method:
Lane Sample Components (µL) 1 1 kb ladder 2 loading dye (5x) + 0.5 ladder + 3.5 MilliQ H2O 2 ECFP 2 DNA + 2 loading dye (5x) + 2 MilliQ H2O 3 EYFP 2 DNA + 2 loading dye (5x) + 2 MilliQ H2O 4 Lumazine 2 DNA + 2 loading dye (5x) + 2 MilliQ H2O
- Ran at 100V for 42 minutes. Stained in EtBr for 15 minutes.
Results: AGAROSE GEL PICTURE
Objective: Ligate dT into pSB1C3.
Method:
- PCR amplify and purify both pSB1C3 and dT
- Restrict both with EcoRI and PstI
- Restrict pSB1C3 with DpnI
- Ligate pSB1C3 and dT
Restriction:
Restriction MilliQ H2O (µL) Buffer Orange (µL) pDNA (µL) Enzyme (µL) pSB1C3 79.25 10 10 0.25 EcoRI + 0.25 PstI + 0.25 DpnI pSB1C3 control 80 10 10 - dT 79.50 10 10 0.25 EcoRI + 0.25 PstI dT control 80 10 10 -
- Restriction were incubated at 37oC for 90 minutes.
- Enzymes were heat killed for 20 minutes at 80oC.
August 3, 2010 Evening
(in lab: KG, JS)
Objective: To run 1.5% agarose of restrictions: pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet
Method: Used a 1.5% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA.
Lane Contents Result 1 1kb ladder 2 pTet (XbaI, EcoRI) good 3 pTet (orange buffer) --- 4 dT (XbaI, EcoR) no good 5 dT (orange buffer) --- 6 mRBS (SpeI, PstI) good 7 mRBS (red buffer) --- 8 sRBS (SpeI, PstI) good 9 sRBS (red buffer) --- 10 mRBS (XbaI, EcoRI) good 11 mRBS (orange buffer) --- 12 sRBS (XbaI, EcoRl) good 13 sRBS (orange buffer) --- 14 pet28(a) good 15 100 bp ladder 16 PSB1C3 not good 17 PSB1C3 restriction digest ---
Lane Contents Result 1 TetR (EcorI, SpeI) good 2 TetR (red buffer) --- 3 pLacI (EcoRI, SpeI) good 4 pLacI (red buffer) --- 5 Mms6 can not tell 6 Mms6 control --- 7 TetR (Xbal, PstI) good 8 TetR (tango buffer) --- 9 pBAD (EcoRI, SpeI) good 10 pBAD (red buffer) --- 11 dT (EcoRI, SpeI) good 12 dT (red buffer) --- 13 pLacI (2) ? 14 dT control not good 15 dT restriction 16 100 bp ladder 17 dT PCR product good 18 Mms6 PCR product good 19 pBAD PCR product good 20 pLacI PCR product good
Objective: To ligate: pBAD-sRBS/mRBS, sRBS/mRBS-TetR, TetR-dT, dT-pTet, pLacI-sRBS/mRBS, Mms6-ptet28(a), dT-PSB1C3
Method: Ligation of Plasmid DNA
15 (µL) pDNA in plasmid, and 15 (µL) of pDNA biobrick)
August 4, 2010
(in lab: JV)
Objective: PCR analysis of ligation product of August 3, 2010
- Ligations
- pBAD-mRBS
- pBAD-SRBS
- SRBS-tetR
- mRBS-TetR
- dt-pTet
- mms6-pET-28a
- dt-pSBIC3
- pLacI-SRBS
- Controls
- pBAD
- TetR
- TetR
- pLacI
- mms6
Method:
PCR: Thermocycler set to iGEM program 7
Component 1X(µL) Master Mix(x16)(µL) Milli-Q H2O 41.8 668.6 10x Pfu Buffer with MgSO4 5 80 dNTPs 1 16 Forward Primer 0.5 8 Reverse Primers 0.5 8 Template DNA 1 16 Pfu polymerase 0.2 3.2
2.5% agarose gel(1x TAE)
lane contents Successful Ligation ? 1 50bp ladder --- 2 dt pSBIC3 --- 3 dt pTet x 4 dt control --- 5 sRBS-TetR x 6 mRBS-TetR ? 7 TetR control --- 8 pLacI-mRBS x 9 pLacI-sRBS ? 10 pLacI control --- 11 Mms6 pET-28a no band 12 Mms6 control --- 13 pBad-SRBS x 14 pBad-mRBS x 15 pBad control ---
- Ran at 100V for 70 minutes.
Results: AGAROSE GEL PICTURE
Objective: Transform the successful ligations
Method: used Competent Cell Transformation protocol
- changes:
- used 50µL aliquottes of DH5&alpha
- did not pipette up and down once, the cells were just swirled 3 times
- added 400µL SOC media, shoock at 370C for 90 min
- platted 250µL and 150µL
Incubated from 12:00AM to 4;00 PM
results contents &250µL 150µL dt-pTet good x - control x x mms6-pET-28a good good dt-pSBIC3 x x mRBS-TetR good good pLacI-mRBS good good SRBS-TetR x x pBAD-SRBS good good + contol good good
August 6/2010 Evening
Objective: Attempt colony pcr for rapid screening
Method: Followed two protocols from openwet
- Knight Protocol
- place 20(µL) sterile H2O in 0.6mL sterile tube
- with P10 pipette set to 3(µL) dip tip into colony
- place pipette tip into water and pipette up and down 20 times(this can be stored at 40C for inoculation of overnight 5mL cultures)
- Endy Protocol
- place 50(µL) sterile H2O in 0.6mL sterile tube
- with PLO pipette (set 3(µL)) dip sterile tip into colony
- place pipette tip into water and pipette up and down 20 times
Knight cont'd Endy cont'd setup 20(µL) reaction setup 20(µL) reaction 1(µL) colony suspension 2(µL) colony suspension 2(µL) 10x p.fu (+Mg SO4 2(µL) 10x p.fu (+Mg SO4 2(µL) dNTP 2(µL) dNTP 1.25(µL)VF2 Primer (10(µM) 0.75(µL)VF2 Primer (10(µM) 1.25(µL)VF Primer (10(µM) 0.75(µL)VF Primer (10(µM) 0.2(µL) Pfu polymerase 0.2(µL) Pfu polymerase 11.8 Milli-Q H2O 12.6 Milli-Q H2O
- as control for each rxn used equal volume of mRBS maxiprep
Knight cont'd Endy cont'd cycling conditions cycling conditions 950C for 15 minutes 950C for 6 minutes *940C for 30 seconds **950C for 30 seconds *560C for 30 seconds **560C for 30 seconds *680C for 1 minutes **700C for 1 minutes 680C for 20 minutes 700C for 10 minutes
(*) were run 39 times
(**) were run 35 times
Made the following program (called COLONYY) Lid preheat 980C
- 980C for 15 minutes
- 980C for 30 seconds
- 560C for 30 seconds
- 68-700C gradient for 1 minute
- 68-700C gradient for 20 minute
- 40C indefinte
bold selections were cycled 39 times
Objective: Analyzed PCR products on 2.5% TAE Agarose gel.
lane sample loaded 1 50bp ladder 1(µL) ladder, 1(µL) dye, 4(µL) H20 2 Knight control 5(µL) sample, 1(µL)dye 3 Knight colony 5(µL) sample, 1(µL)dye 4 Endy control 5(µL) sample, 1(µL)dye 5 Endy colony 5(µL) sample, 1(µL)dye 6 50bp ladder 1(µL) ladder, 1(µL) dye, 4(µL) H20 7 lumazine(justin's) 5(µL) sample, 1(µL)dye
Repeat gel with template controls
lane sample loaded 1 50bp ladder 0.5(µL) ladder, 2(µL) dye, 9.5(µL) H20 2 Endy Template 5(µL) colony suspension, 1(µL)dye, 4(µL) H20 3 Endy mRBS Control (PLR) 5(µL) sample, 1(µL)dye 4 Endy mRBS-TetR colony(PCR) 5(µL) sample, 1(µL)dye 4 mRBS template 0.5(µL) sample, 1(µL)dye, 4(µL) H20 5 Knight Template 0.25(µL) colony suspension, 1(µL)dye, 4(µL) H20 6 Knight mRBS control 5(µL) ladder, 1(µL) dye 7 Knight-mRBS-TetR colony 5(µL) sample, 1(µL)dye 1 1Kb ladder 0.5(µL) ladder, 2(µL) dye, 9.5(µL) H20
- ran at 100V for 75 minutes
Aug 9/2010
(In Lab: AV)
Objective: Prepared glycerol stocks & Miniprepped the following using the Qiagen Protocol.
pLacI-mRBS Colony 1 pLacI-mRBS Colony 2 pLacI-sRBS Colony 2 pLacI-sRBS Colony 3 pBAD-mRBS Colony 1 pBAD-mRBS Colony 2 pBAD-sRBS Colony 1 pBAD-sRBS Colony 2 dT-pTet Colony 1 dT-pTet Colony 3 mRBS-TetR Colony 1 mRBS-TetR Colony 3
Objective: To determine which of the previous ligations worked.
Method: Restricted with single cutter and double cutter.
Restriction Reaction (SINGLE)
Ingredient Volume(µL) MilliQ H20 Water 15.75 Orange Buffer (10x) 2 pDNA 2 EcoRI 0.25
Restriction Reaction (DOUBLE)
Ingredient Volume(µL) MilliQ H20 Water 15.50 Orange Buffer (10x) 2 pDNA 2 EcoRI 0.25 PstI 0.25
Unrestricted Control
Ingredient Volume(µL) MilliQ H20 Water 16 Orange Buffer (10x) 2 pDNA 2
DNA was restricted for 1 hour at 37oC.
Analyzed results on a 2% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA. Load order as follows:
Lane Contents 1 1kb ladder 2 50 bp ladder 3 dT-pTet 1 DRD 4 dT-pTet 1 SRD 5 dT-pTet 1 URD 6 dT-pTet 3 DRD 7 dT-pTet 3 SRD 8 dT-pTet 3 URD 9 pLacI-mRBS 1 DRD 10 pLacI-mRBS 1 SRD 11 pLacI-mRBS 1 URD 12 pLacI-mRBS 2 DRD 13 pLacI-mRBS 2 SRD 14 pLacI-mRBS 2 URD 15 pLacI-sRBS 1 DRD 16 pLacI-sRBS 1 SRD 17 pLacI-sRBS 1 URD 18 pLacI-sRBS 3 DRD 19 pLacI-sRBS 3 SRD 20 pLacI-sRBS 3 URD
Lane Contents 1 1 kb ladder 2 50 bp ladder 3 pBAD-mRBS 1 DRD 4 pBAD-mRBS 1 SRD 5 pBAD-mRBS 1 URD 6 pBAD-mRBS 2 DRD 7 pBAD-mRBS 2 SRD 8 pBAD-mRBS 2 URD 9 pBAD-sRBS 1 DRD 10 pBAD-sRBS 1 SRD 11 pBAD-sRBS 1 URD 12 pBAD-sRBS 2DRD 13 pBAD-sRBS 2 SRD 14 pBAD-sRBS 2 URD 15 mRBS-TetR 1 DRD 16 mRBS-TetR 1 SRD 17 mRBS-TetR 1 URD 18 mRBS-TetR 3 DRD 19 mRBS-TetR 3 SRD 20 mRBS-TetR 3 URD
GEL PICTURE!
Aug 9/2010 Evening
(In Lab: JV, AS)
Objective: To ligate: lumazine into vector upstream of dT. Lumazine and mms6 into pET28a.
Method:
- Restrictions
Set up reactions as follows:
- Restrict Lumazine wit EcoRI and SpeI (Red Buffer)
- Restrict the dT with XbaI and EcoRI (Orange Buffer)
- Restrict Lumazine Synthase with NotI (Red Buffer)
Component Volume (µL) MilliQ H2O 15.6 or 15.8 Buffer 2 pDNA 2 Enzyme 0.20 + 0.20 Incubated reactions for 60 minutes at 37oC
- Ligation
Reaction set up as follows:Incubated reactions overnight at room temperature.
- T4 DNA ligase - 0.25µL
- DNA 1 - 8µL
- DNA 2 - 8µL
- 10x Ligation Buffer - 2µL
- MilliQ H2O - 1.75µL
Aug 10/2010
(In Lab: JV)
Objective: Reran large gel from Aug 9/2010.
Load order was as follows:
Lane Contents 1 50 bp ladder 2 pBAD-mRBS 2 URD 3 pBAD-mRBS 2 SRD 4 pBAD-mRBS 2 DRD 5 pLacI-sRBS 3 URD 6 pLacI-sRBS 3 SRD 7 pLacI-sRBS 3 DRD 8 pLacI-mRBS 2 URD 9 pLacI-mRBS 2 SRD 10 pLacI-mRBS 1 DRD 11 dT-pTet 3 URD 12 dT-pTet 3 SRD 13 dT-pTet 3 DRD 14 pBAD-mRBS 1 URD 15 pBAD-mRBS 1 SRD 16 pBAD-mRBS 1 DRD 17 pLacI-sRBS 2 URD 18 pLacI-sRBS 2 SRD 19 pLacI-sRBS 2 DRD 20 1 kb ladder
Lane Contents 1 50 bp ladder 2 pLacI-mRBS 1 URD 3 pLacI-mRBS 1 SRD 4 pLacI-mRBS 1 DRD 5 dT-pTet 1 URD 6 dT-pTet 1 SRD 7 dT-pTet 1 DRD 8 mRBS-TetR 3 URD 9 mRBS-TetR 3 SRD 10 mRBS-TetR 3 DRD 11 mRBS-TetR 1 URD 12 mRBS-TetR 1 SRD 13 mRBS-TetR 1 DRD 14 pBAD-sRBS 2 URD 15 pBAD-sRBS 2 SRD 16 pBAD-sRBS 2 DRD 17 pBAD-sRBS 1 URD 18 pBAD-sRBS 1 SRD 19 pBAD-sRBS 1 DRD 20 1 kb ladder
GEL PICTURE!
Aug 13/2010
(In Lab: AS)
Objective: PCR amplify minipreps prepared on Aug 9/2010 to screen for properly assembled BioBricks.
Method:
PCR: Thermocycler set to iGEM program 7
Component 1X(µL) Master Mix(x12.5)(µL) Milli-Q H2O 41.85 523.1 10x Pfu Buffer with MgSO4 5 62.5 dNTPs 1 12.5 Forward Primer (VF2) 0.5 6.25 Reverse Primers (VR) 0.5 6.25 Template DNA 1 Pfu polymerase 0.15 1.888
Added 49µL Master Mix to each reaction tube.
Aug 14/2010
(In Lab: AS)
2.5% agarose gel(1x TAE)
lane contents 1 pBAD-mRBS 1 2 pBAD-mRBS 2 3 pBAD-sRBS 1 4 pBAD-sRBS 2 5 mRBS-TetR 1 6 mRBS-TetR 3 7 50 bp Ladder 8 dT-pTet 1 9 dT-pTet 3 10 pLacI-mRBS 1 11 pLacI-mRBS 2 12 pLacI-sRBS 2 13 pLacI-sRBS 3 14 MT 15 MT 16 MT 17 MT 18 MT 19 MT 20 MT 21 No Lanes 22 No Lanes 23 No Lanes 24 No Lanes 25 No Lanes 26 No Lanes 27 No Lanes
lane contents 1 K249001 2 K249004 3 K249005 4 K249006 5 MT 6 K249008 7 K249008 (Qiagen) 8 K249014 9 K249017 10 50 bp Ladder 11 1 12 2 13 3 14 4 15 5 16 xylE-dT 17 Lumazine-dT 18 pLacI-sRBS 19 MT 20 MT 21 MT 22 MT 23 MT 24 MT 25 MT 26 MT 27 MT GEL PICTURE!
Aug 14/2010 Evening
(In Lab: AS)