Team:Lethbridge/Notebook/Lab Work/July

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==July 8/2010 - Evening==
==July 8/2010 - Evening==
(In lab: KG)<br>
(In lab: KG)<br>
-
<b>Objective:</b>To transform TetR in in pSB1A2 plasmid (BBa_C0040) into DH5&alpha; from 2010 iGEM Distribution Kit Plate Well 4A<br>
+
<b>Objective:</b>To transform TetR in in pSB1A2 plasmid (BBa_C0040) into DH5&alpha; from 2010 iGEM Distribution Kit Plate Well 4A using the Competent Cell Transformation Protocol.</br>
<b>Method:</b>Transformed pUC19 as a positive control.</br>
<b>Method:</b>Transformed pUC19 as a positive control.</br>

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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:

LaneSampleComponents (µL)
11kb Ladder0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O
21 - pBAD (A4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
32 - pBAD (A5)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
43 - SRBS (A6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
54 - SRBS (A7)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
65 - CFP Complete (A8)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
76 - SRBS (A10)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
87 - EYFP (B1)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
98 - N term tag (B2)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
109 - pSB NEYFP (B4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1110 - pSB NEYFP (B5)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1211 - CFP (B6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1312 - pBAD-TetR (B10)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1413 - D31 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1514 - C term (D4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1615 - C term (D5)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1716 - pLacI (D6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1817 - NEYFP (E2)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1918 - CEYFP (E6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
2019 - CEYFP (E7)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
11kb ladder0.5 Ladder + 2 Dye (6X) + 7.5 Milli-Q H2O
220 - EYFP (E8)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
321 - EYFP (E9)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
422 - EYFP (E10)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
523 - ECFP (F1)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
624 - ECFP (F2)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
725 - ECFP (F3)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
826 - pBAD-TetR (F4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
927 - pBAD-TetR (F5)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1028 - EYFP (G1)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1129 - pSB CEYFP (G4)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1230 - pBAD (1) (G6)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1331 - pBAD (2) (G7)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1432 - N term tag (G8)1 DNA + 2 Dye (6X) + 7 Milli-Q H2O
1533 - 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)
00.071
10.390
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:

ComponentVolume (µL)
DNA0.071
Buffer Orange0.390
NotI0.606
MilliQ H2O1.250

Incubated for 1 hour at 37oC. Heat killed on heat block at 80oC for 20 mins.</br>

2) 1% Agarose gel

LaneSampleVolume (µL)
11 kb Ladder0.5 Ladder + 2 Loading dye (6x) + 7.5 MilliQ H2O
2PET28a restricted8 DNA + 2 Loading dye (6x)
3PET28a8 DNA + 2 Loading dye (6x)
4Lumazine restricted8 DNA + 2 Loading dye (6x)
5Lumazine8 DNA + 2 Loading dye (6x)
6mms6 restricted8 DNA + 2 Loading dye (6x)
7mms68 DNA + 2 Loading dye (6x)
8xylE restricted8 DNA + 2 Loading dye (6x)
9xylE8 DNA + 2 Loading dye (6x)
10EmptyEmpty

Ran at 100V for 80 mins.</br>

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:

July 8/2010 - Evening

(In lab: KG)
Objective:To transform TetR in in pSB1A2 plasmid (BBa_C0040) into DH5α from 2010 iGEM Distribution Kit Plate Well 4A using the Competent Cell Transformation Protocol.</br>

Method:Transformed pUC19 as a positive control.</br>
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