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Team:Lethbridge/Notebook/Lab Work/July
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Back to Lab Work
July 2010
July 5/2010
(In Lab: JV, AV, HB)
Objective: Run a 1% agarose gel of purified PCR samples from June 24/10
Method:
| Lane | Sample | Components (µL) |
| 1 | 1kb Ladder | 0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O |
| 2 | 1 - pBAD (A4) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 3 | 2 - pBAD (A5) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 4 | 3 - SRBS (A6) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 5 | 4 - SRBS (A7) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 6 | 5 - CFP Complete (A8) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 7 | 6 - SRBS (A10) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 8 | 7 - EYFP (B1) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 9 | 8 - N term tag (B2) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 10 | 9 - pSB NEYFP (B4) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 11 | 10 - pSB NEYFP (B5) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 12 | 11 - CFP (B6) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 13 | 12 - pBAD-TetR (B10) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 14 | 13 - D3 | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 15 | 14 - C term (D4) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 16 | 15 - C term (D5) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 17 | 16 - pLacI (D6) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 18 | 17 - NEYFP (E2) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 19 | 18 - CEYFP (E6) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 20 | 19 - CEYFP (E7) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 1 | 1kb ladder | 0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O |
| 2 | 20 - EYFP (E8) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 3 | 21 - EYFP (E9) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 4 | 22 - EYFP (E10) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 5 | 23 - ECFP (F1) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 6 | 24 - ECFP (F2) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 7 | 25 - ECFP (F3) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 8 | 26 - pBAD-TetR (F4) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 9 | 27 - pBAD-TetR (F5) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 10 | 28 - EYFP (G1) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 11 | 29 - pSB CEYFP (G4) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 12 | 30 - pBAD (1) (G6) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 13 | 31 - pBAD (2) (G7) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 14 | 32 - N term tag (G8) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
| 15 | 33 - lumazine (G9) | 1 DNA + 2 Dye (6X) + 7 Milli-Q H2O |
Ran gel at 100V for 45 minutes.
Picture to come!!!!!!!!!
July 5/2010 Evening
Objective: To over-express CFP complete in DH5α
Method:
1) Inoculated 6mL culture with ampicillin and glycerol stock A6-May 13,2010 CFP complete 2) Went in the shaker at 6:50pm
July 6/2010
(In lab: JV, AV, HB)
Objective: To continue the over-expression of CFP complete in DH5α
Method:
1) Put both cultures (taken out of shaker at 9:15am) and put them in 500mL of LB w/ Amp.
2) initial OD was 0.071 (600λ)
Issue:
- After checking the the sequencing it was evident that our promotor is always off. It is turned off by the product of the gene TetR. Which is not part of our construct.
| Time (hours) | OD (600λ) |
| 0 | 0.071 |
| 1 | 0.390 |
| 1.5(T0) | 0.606 |
| 2.5 (T1) | 1.250 |
| 3.5(T2) | 3.04 |
| 4.5(T3) | 2.75 |
Results: Ran samples on a 15% SDS-page. The gel did not show any signs of over-expression.
Objective: To determine if maxipreps were successful via restriction by NotI and running on 1% Agarose gel
Method:
1) Restriction:
| Component | Volume (µL) |
| DNA | 0.071 |
| Buffer Orange | 0.390 |
| NotI | 0.606 |
| MilliQ H2O | 1.250 |
Incubated for 1 hour at 37oC.
Heat killed on heat block at 80oC for 20 mins.
2) 1% Agarose gel
| Lane | Sample | Volume (µL) |
| 1 | 1 kb Ladder | 0.5 Ladder + 2 Loading dye (6x) + 7.5 MilliQ H2O |
| 2 | PET28a restricted | 8 DNA + 2 Loading dye (6x) |
| 3 | PET28a | 8 DNA + 2 Loading dye (6x) |
| 4 | Lumazine restricted | 8 DNA + 2 Loading dye (6x) |
| 5 | Lumazine | 8 DNA + 2 Loading dye (6x) |
| 6 | mms6 restricted | 8 DNA + 2 Loading dye (6x) |
| 7 | mms6 | 8 DNA + 2 Loading dye (6x) |
| 8 | xylE restricted | 8 DNA + 2 Loading dye (6x) |
| 9 | xylE | 8 DNA + 2 Loading dye (6x) |
| 10 | Empty | Empty |
Ran at 100V for 80 mins.
Picture to come!!!!!!!!!
More to fill in, but Anthony does not understand the stuff written in the lab book
July 8/2010
(In lab: JV, AV, HB, HS)
Objective:
Anthony does not understand the stuff written in the lab book.
July 8/2010 - Evening
(In lab: KG)
Objective:To transform TetR in pSB1A2 plasmid (BBa_C0040 - 2010 iGEM Distribution Kit Plate Well 4A) and pTetR in pSB1A2 plasmid (BBa_R0040 - 2010 iGEM Distribution Kit Plate Well 6) into DH5α.
Method:Used the Competent Cell Transformation Protocol as well as transformed pUC19 as a positive control.
Results:
| Plate | Number of Colonies |
| 50 µL TetR | 0 |
| 200 µL TetR | 5 |
| 50 µL pTetR | 4 |
| 200 µL pTetR | 34 |
| 50 µL pUC19 | 3 |
| 200 µL pUC19 | 4 |
July 9/2010
(In lab: JV)
Objective:To overexpress pLacI-mRBS-mms6-dT construct.
Method:Used the Overexpression Protocol.
| Time (hours) | OD (600λ) |
| 0 | 0.052 |
| 1 | 0.111 |
| 2 | 0.315 |
| 2.5 | 0.449 |
| 3(T0) | 0.772 |
| 4(T1) | 2.22 |
| 5(T2) | 2.06 |
| 6(T3) | 2.50 |
Samples were run on a 15% SDS PAGE for 20 minutes at 80V and 1 hour at 200V.
SDS PAGE picture!!!!!!!!!!!
July 10/2010
(In lab: JV)
Objective:To determine if maxipreps finished on July 9th and 10th have significant concentrations of DNA.
Method:Run 1% Agarose gel
| Lane | Sample | Components (µL) |
| 1 | dT | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
| 2 | mms6 (1) | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
| 3 | mms6 (2) | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
| 4 | pBAD-TetR (1) | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
| 5 | pBAD-TetR (2) | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
| 6 | mRBS | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
| 7 | pLacI | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
| 8 | sRBS | 2 DNA + 2 loading dye (6x) + 6 MilliQ H2O |
| 9 | 1 kb Ladder | 0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H2O |
| 10 | Empty | Empty |
Ran at 100V for 40 minutes. Strained in EtBr for 10 minutes.
AGAROSE GEL PICTURE
July 12/2010
(In lab: AV,HB,JV)
Objective: Maxiprep pLacI (A2) from glycerol stock.
Method: Used Maxiprep Protocol. Cell pellet weighed 1.02g.
Objective: Add dT to the end of mms6, xylE, and lumazine.
Method:
1) Restrict "Part 1" BioBricks: mms6, xylE, and lumazine with EcoRI and SpeI.
| Component | Volume (µL) |
| pDNA | 2 |
| Red Buffer | 2 |
| EcoRI | 0.25 |
| SpeI | 0.25 |
| MilliQ H2O | 15.5 |
2) Restrict "Part 2" BioBrick: dT with EcoRI and XbaI.
| Component | Volume (µL) | 2.5x Volume (µL) |
| pDNA | 2 | 5 |
| Orange Buffer | 2 | 5 |
| EcoRI | 0.25 | 0.625 |
| XbaI | 0.25 | 0.625 |
| MilliQ H2O | 15.5 | 38.75 |
3) 1% Agarose gel (1x TAE) of restricted DNA
| Lane | Sample | Component (µL) |
| 1 | 1 kb Ladder | 0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H2O |
| 2 | dT | 8 DNA + 2 loading dye (6x) |
| 3 | dT restricted | 8 DNA + 2 loading dye (6x) |
| 4 | mms6 | 8 DNA + 2 loading dye (6x) |
| 5 | mms6 restricted | 8 DNA + 2 loading dye (6x) |
| 6 | xylE | 8 DNA + 2 loading dye (6x) |
| 7 | xylE restricted | 8 DNA + 2 loading dye (6x) |
| 8 | Lumazine | 8 DNA + 2 loading dye (6x) |
| 9 | Lumazine restricted | 8 DNA + 2 loading dye (6x) |
| 10 | Empty | Empty |
Ran at 100V for 43 minutes. Stained in EtBr for 10 minutes.
AGAROSE GEL PICTURE
4) Ligate restricted dT to the ends of the "Part 1" Biobricks.
July 12/2010 - Evening
(In lab: KG,TF)
Objective: Continue with addition of dT to the ends of mms6, xylE, and lumazine.
Method:
4) Ligate restricted dT to the ends of the "Part 1" Biobricks.
dT to mms6:
| Component | Volume (µL) |
| T4 DNA Ligase | 0.25 |
| 10x T4 Ligation Buffer | 2 |
| dT | 8.3 |
| mms6 | 9.2 |
| MilliQ H2O | 0.25 |
dT to xylE:
| Component | Volume (µL) |
| T4 DNA Ligase | 0.25 |
| 10x T4 Ligation Buffer | 2 |
| dT | 8.3 |
| xylE | 3.4 |
| MilliQ H2O | 6.05 |
dT to lumazine:
| Component | Volume (µL) |
| T4 DNA Ligase | 0.25 |
| 10x T4 Ligation Buffer | 2 |
| dT | 8.3 |
| lumazine | 3.35 |
| MilliQ H2O | 6.1 |
Ligations were incubated at room temperature overnight.
Objective: Prepare mastermix for four PCR reactions for following day.
Method:
| Component | Volume (µL) | 5x Volume (µL) |
| 10mM dNTPs | 1 | 5 |
| 5x Phusion Buffer | 4 | 20 |
| Forward Primer (VF2) | 1 | 5 |
| Reverse Primer (VR) | 1 | 5 |
| MilliQ H2O | 10.8 | 54 |
July 13/2010
(in lab: JV)
Objective: To determine if ligations of previous day (July 12/2010) were successful.
Method:
1) Use prepared mastermix to run a PCR of ligated mms6-dT, ligated xylE-dT, ligated lumazine-dT, and control-dT
- To each PCR tube, add 17.8&mirco;L of mastermix, 0.2µL of Phusion DNA polymerase, and 2µL of template DNA.
- Ran PCR's for 36 cycles using the iGEM preset.
2) 2% Agarose gel
| Lane | Sample | Components (µL) |
| 1 | 50 bp Ladder | 1 ladder + 2 loading dye (6x) + 7 MilliQ H2O |
| 2 | mms6-dT | 8 PCR product + 2 loading dye (6x) |
| 3 | xylE-dT | 8 PCR product + 2 loading dye (6x) |
| 4 | lumazine-dT | 8 PCR product + 2 loading dye (6x) |
| 5 | dT | 8 PCR product + 2 loading dye (6x) |
| 6 | Empty | Empty |
| 7 | Empty | Empty |
| 8 | Empty | Empty |
| 9 | Empty | Empty |
| 10 | Empty | Empty |
Ran at 100V for __ minutes. Stained in EtBr for 10 minutes.
AGAROSE GEL PICTURE
July 14/2010
(in lab:J.S, K.G )
Objective: Inoculate culture for maxi prep with placI
Method: To a 450mL solution of LB media 4.5µL of ampicillin was added with glycerol placI aseptically.
(in lab:J.S, K.G )
Objective: Restriction of dT and mms6
protocol
1)
| Component | Volume (µL) |
| Buffer&* | 2 |
| 10x T4 Milli Q H2O | 15.5 |
| pDNA** | 2 |
| Restriction enzymes*** | 0.25 |
* Orange buffer was used for dT, and Red buffer was used for mms6
**pDNA was dT and mms6
***Restriction enzymes for dT were XbalI and EcoRI, and for mms6 SpeI and EcoRI
2) Reaction was incubated at 370C for 1 hour. Start 8:50pm till 9:50pm After incubation reaction was heat shocked at 800C for 20 minutes
(in lab:J.S, K.G )
Objective: Ligation of dT to each of mms6, xylE and lumazine.
protocol
1)
dT to mms6:
| Component | Volume (µL) |
| T4 DNA Ligase | 0.25 |
| 10x T4 Ligation Buffer | 2 |
| dT | 8.3 |
| mms6 | 9.2 |
| MilliQ H2O | 0.25 |
dT to xylE:
| Component | Volume (µL) |
| T4 DNA Ligase | 0.25 |
| 10x T4 Ligation Buffer | 2 |
| dT | 8.3 |
| xylE | 3.4 |
| MilliQ H2O | 6.05 |
dT to lumazine:
| Component | Volume (µL) |
| T4 DNA Ligase | 0.25 |
| 10x T4 Ligation Buffer | 2 |
| dT | 8.3 |
| lumazine | 3.35 |
| MilliQ H2O | 6.1 |
2)
Incubated reaction overnight at room temperature
July 15/2010
(in lab: AV)
Objective: Maxiprep pLacI and mms6.
| component | pallet weight (g) |
| mms6 | 1.02 |
| pLacI | 1.54 |
July 15/2010 Evening
(in lab: AV)
Objective: transform ligations from July 14 and July 12,2010;xylE/dt, mms6/dt lumazine/dt into DH5&alpha.
Also to transform mms6 from July 6 and July 10 into Bl21(DE3)
Protocol: to transform competent cells see protocol:[1]
* cells were incubated on ice for 30 minutes started at 7:00-7:30. **incubated for 1 hour from 8:00 till 9:00
| Results | ||
| plate | # of colonies | Components (µL) |
| 1 | 200µL xylE-dt July 12 | 0 |
| 2 | 200µL mms6-dt July 12 | 1 |
| 3 | 200µL lumazine-dt | 1 |
| 4 | 200µL mms6 maxiprep July 6 | Lawn |
| 5 | 200µL positve control puC19 into BL21(DE3) | Lawn |
| 6 | 200µL xylE-dt July 14 | 0 |
| 7 | 200µL mms6-dt July 14 | 120 |
| 8 | 200µL lumazine-dt July14 | 0 |
| 9 | 200µL mms6 maxiprep July 10 | . |
*because of a shortage of plates not all transformations were plated at 50µL and 200µL
July 16,2010
(in lab: M.C, D.M)
Objective: PCR amplify mms6-dT, xylE-dT, lumazine-dT legations along with dT maxi prep for comparison
Protocol:
Master Mix
| Component | Volume (µL) |
| Milli-Q H2O< | 54 |
| Pfu Buffer + MgSO4 | 20 |
| 10 mM dMTPs | 5 |
| forward primer | 5 |
| reverse primer | 5 |
89 µL TOTAL--> 17.8 into each PCR reaction
+ 2 µL ligation + 2 µL Pfu polymerase
Ran iGem-ligTest in thermocycler
July 19, 2010
(in lab: A.V, H.B)
Objective: Purification pf pLacI and mms6 maxipreps done on July 14 and 16, 2010 using the biobasic protocol for purification of PCR products.
Objective: Add pLacI to sRBS and add dT to xylE and lumazine
Method:
Restrict pLacI, xylE and lumazine with SpeI and EcoRI and restrict dT and sRBS with EcorI and XbaI
1)Restrictions:
pLacI, xylE, and Lumazine
| Component | Volume (µL) |
| Milli-Q H2O | 15.5 |
| Red Buffer4 | 2 |
| SpeI | 0.25 |
| EcoRI | 0.25 |
| pDNA | 2 |
dT
| Component | Volume (µL) |
| Milli-Q H2O | 38.75 |
| Orange Buffer4 | 5 |
| XbaI | 0.625 |
| EcoRI | 0.625 |
| pDNA | 5 |
sRBS
| Component | Volume (µL) |
| Milli-Q H2O | 15.5 |
| Red Buffer4 | 2 |
| xBal | 0.25 |
| EcoRI | 0.25 |
| pDNA | 2 |
Restriction incubation at 37.50C started at 11:55am. Ended at 12:55pm Heat killed enzymes at 800C for 20 minutes
2)Restrictions were run on a 1% agarose gel (1 X TAE)
1% Agarose gel
| Lane | Sample | Components (µL) |
| 1 | 1kB Ladder | 0.5 ladder + 2 loading dye (5x) + 5.5 MilliQ H2O |
| 2 | pLacI | 6 pDNA + 2 loading dye (5x) |
| 3 | pLacI restriction digest | 6 pDNA + 2 loading dye (5x) |
| 4 | sRBS | 6 pDNA + 2 loading dye (5x) |
| 5 | sRBS restriction digest | 6 pDNA + 2 loading dye (5x) |
| 6 | xylE | 6 pDNA + 2 loading dye (5x) |
| 7 | xylE restriction digest | 6 pDNA + 2 loading dye (5x) |
| 8 | lumazine | 6 pDNA + 2 loading dye (5x) |
| 9 | lumazine restriction digest | 6 pDNA + 2 loading dye (5x) |
| 10 | dT | 6 pDNA + 2 loading dye (5x) |
| 11 | dT restriction digest | 6 pDNA + 2 loading dye (5x) |
Gel ran for 70 minutes at 100V and was stained in EtBr for 10 minutes
AGAROSE GEL PICTURE
Results after quantifying restriction digest
| Lane | Content | Quantity of DNA (ng/µL) |
| 1 | 1kB Ladder | 12.5 |
| 3 | pLacI restriction digest | 5.21 |
| 5 | sRBS restriction digest | 3.72 |
| 7 | xylErestriction digest | 3.36 |
| 9 | Lumazine restriction digest | 2.63 |
| 11 | dT restriction digest | 2.98 |
July 19, 2010 Evening
(K.G)
Objective: Ligate together rbs-xylE and dT, lumazine and dT, also pLacI and sRBS.
Method: All ligation mixes had:
- 10X T4 Ligation Buffer
- Milli-Q H2O to fill to 20µL
- T4 DNA Ligase
- 20ng plasmid DNA
Ligations were left over-night at room temperature.
(J.V.)
Objective:Determine the results of the transformations done by K.G. on July 15/2010.
Method: Inoculate 5mL LB media w/Amp and colony. Incubated over night at 37oC.
Results: Lumazine Synthase with dT ligated onto it grew.
July 20, 2010
(AV, HB)
Objective:Miniprep lumazine-dt and 4 N-terminus tags and analyze.
Method:
- Use boiling lysis miniprep to isolate plasmid DNA.
- PCR amplify BioBrick part to determine if the correct DNA was isolated.
- Ran a 1% Agarose gel to visualize PCR products.
