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Team:Lethbridge/Notebook/Lab Work/July
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| - | Ran at 100V for 40 minutes. Strained in EtBr for 10 minutes. | + | Ran at 100V for 40 minutes. Strained in EtBr for 10 minutes.<br> |
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| + | <font color ="red">AGAROSE GEL PICTURE</font> | ||
==July 12/2010== | ==July 12/2010== | ||
(In lab: AV,HB,JV)<br> | (In lab: AV,HB,JV)<br> | ||
<b>Objective:</b> | <b>Objective:</b> | ||
Revision as of 18:45, 26 July 2010
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Back to Notebook
Back to Lab Work
Contents |
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)
