From 2010.igem.org

(Difference between revisions)

Revision as of 18:41, 26 July 2010

[http://2010.igem.org/wiki/index.php?title=Team:Lethbridge&Home ]	[http://2010.igem.org/Team:Lethbridge/Team ]	[http://2010.igem.org/Team:Lethbridge/Project ]	[http://2010.igem.org/Team:Lethbridge/Notebook ]	[http://2010.igem.org/Team:Lethbridge/Parts ]	[http://2010.igem.org/Team:Lethbridge/Modeling ]	[http://2010.igem.org/Team:Lethbridge/Ethics ]	[http://2010.igem.org/Team:Lethbridge/Safety ]

Back to Notebook
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 37^oC. Heat killed on heat block at 80^oC 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:

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.

July 12/2010

(In lab: AV,HB,JV)

Objective:

@@ Line 192: / Line 192: @@
 ==July 9/2010==
 (In lab: JV)<br>
-<b>Objective:</b>To overexpress pLacI-mRBS-mms6-dT construct.</br>
+<b>Objective:</b>To overexpress pLacI-mRBS-mms6-dT construct.<br>
-<b>Method:</b>Used the Overexpression Protocol.</br>
+<b>Method:</b>Used the Overexpression Protocol.<br>
 <table><table border ="3">
 <tr><td><b>Time (hours)</b></td><td><b>OD (600&lambda;)</b></td></tr>
@@ Line 207: / Line 207: @@
 </table><br>
-Samples were run on a 15% SDS PAGE for 20 minutes at 80V and 1 hour at 200V.</br>
+Samples were run on a 15% SDS PAGE for 20 minutes at 80V and 1 hour at 200V.<br>
 <font color ="red">SDS PAGE picture!!!!!!!!!!!</font>
+==July 10/2010==
+(In lab: JV)<br>
+<b>Objective:</b>To determine if maxipreps finished on July 9th and 10th have significant concentrations of DNA.<br>
+<b>Method:</b>Run 1% Agarose gel<br>
+<table><table border ="3">
+<tr><td><b>Lane</b></td><td><b>Sample</b></td><td><b>Components (&micro;L)</b></td></tr>
+<tr><td>1<td>dT</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr>
+<tr><td>2<td>mms6 (1)</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr>
+<tr><td>3<td>mms6 (2)</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr>
+<tr><td>4<td>pBAD-TetR (1)</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr>
+<tr><td>5<td>pBAD-TetR (2)</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr>
+<tr><td>6<td>mRBS</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr>
+<tr><td>7<td>pLacI</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr>
+<tr><td>8<td>sRBS</td><td>2 DNA + 2 loading dye (6x) + 6 MilliQ H<sub>2</sub>O</td></tr>
+<tr><td>9<td>1 kb Ladder</td><td>0.5 ladder + 2 loading dye (6x) + 7.5 MilliQ H<sub>2</sub>O</td></tr>
+<tr><td>10<td>Empty</td><td>Empty</td></tr>
+</table><br>
+Ran at 100V for 40 minutes. Strained in EtBr for 10 minutes.
+==July 12/2010==
+(In lab: AV,HB,JV)<br>
+<b>Objective:</b>

Team:Lethbridge/Notebook/Lab Work/July