| 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
In Lab: JV
Objective: Put lumazine synthase gene and mms6 into pET-28(A) for future overexpression.
Method: Restrict mms6 from Mr. Gene with NotI. Large quantities of DNA can be used so a gel extraction of the part can be done. PCR lumazine synthase out of its backbone. Restrict off the extra DNA fragments from the PCR with NotI. Restrict pET-28A with notI. Ligate.
Restriction:
| Restriction | MilliQ H2O (µL) | Buffer Orange (µL) | pDNA (µL) | Enzyme (µL) |
| mms6 | 799.5 | 100 | 100 | 1 NotI |
PCR:
| Component | 1X(µL)
|
| Milli-Q H2O | 41.85
|
| 10x Pfu Buffer with MgSO4 | 5
|
| dNTPs | 1
|
| Forward Primer (VF2) | 0.5
|
| Reverse Primers (VR) | 0.5
|
| Template DNA (Lumazine Synthase) | 1
|
| Pfu polymerase | 0.2
|
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
- Restrict Lumazine wit EcoRI and SpeI (Red Buffer)
- Restrict the dT with XbaI and EcoRI (Orange Buffer)
- Restrict Lumazine Synthase with NotI (Red Buffer)
Set up reactions as follows:
| 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:
- T4 DNA ligase - 0.25µL
- DNA 1 - 8µL
- DNA 2 - 8µL
- 10x Ligation Buffer - 2µL
- MilliQ H2O - 1.75µL
Incubated reactions overnight at room temperature.
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)
"
