Team:Lethbridge/Notebook/Lab Work/September

From 2010.igem.org

(Difference between revisions)

Revision as of 05:33, 4 October 2010

Feel free to look around our notebook!

Here you can check out the work we have done in the lab, click on a month to take a look!

Contents

1 September

1.1 September 2, 2010

1.1.1 ADS

1.2 September 14, 2010

1.2.1 JF

1.3 September 14, 2010

1.3.1 ADS

1.4 September 20, 2010

1.4.1 ADS

1.5 September 21, 2010

1.5.1 ADS

1.6 September 21, 2010

1.6.1 ADS

1.7 September 23, 2010

1.7.1 JV

1.7.2 ADS

1.8 September 24, 2010

1.8.1 ADS

1.9 September 25, 2010

1.9.1 JV

September

September 2, 2010

ADS

Objective: Assemble lumazine-dT into pSB1C3 using NEB BioBrick Assembly Kit.
Method: Have the following parts: dT [28 ng/(µL)]; lumazine [78 ng/(µL)]; psB1C3 [14 ng/(µL)].

Require 500 ng DNA each part in a 50(µL) rxn therefore 17.9(µL) dT and 6.4(µL) Lumazine. Need 250 ng pSB1C3 or 17.9(µL)

Restriction Reactions -

Ingredient Volume(µL)

MilliQ H₂0 Water 36.1

NEBuffer 2 (10x) 5

Upstream Part (Lumazine) 26.4

EcoRI-HF 1

SpeI 1

100X BSA 0.5

Ingredient Volume(µL)

MilliQ H₂0 Water 24.6

NEBuffer 2 (10x) 5

Downstream Part (dT) 17.9

XbaI 1

PstI 1

100X BSA 0.5

Ingredient Volume(µL)

MilliQ H₂0 Water 3.35

NEBuffer 2 (10x) 2.5

Destination Plasmid (pSB1C3) 17.9

EcoRI-HF .5

PstI .5

100X BSA 0.25

2(µL) was removed prior to adding enzymes for analysis on gel.
Split digestion reactions in half: dT - 2 @ 24(µL); Lum - 2 @ 24(µL); psB1C3 - 2 @ 11.5(µL)
Ran 10 minute and 60 minute reactions at 37^oC. Followed by 20 minute heat shock at 80^oC.
2(µL) was removed from the heat killed reactions for analysis on gel.

Performed 4 combinations of ligation: 10 min Restriction + 10 min Ligation; 10 min Restriction + Overnight Ligation; 60 min Restriction + 10 min Ligation; 60 min Restriction + Overnight Ligation

Ingredient 1X(µL) 2X Master Mix(x2.5)(µL)
MilliQ H₂0 Water 11 77.5
T4 Ligase Buffer (10x) 2 5
Upstream Part 2 5
Downstream Part 2 5
T4 DNA Ligase 1 2.5
Plasmid Part 2 5

Incubated 10 minute and overnight ligations at room temperature ( 25^oC). Heat killed ligase at 80^oC for 20 min.

2(µL) of each reaction was taken for analysis on gel.

Confirmed ligation with PCR analysis. Analyzed restriction, ligation and PCR on a 1.5% TAE agarose gel.

Component 1X(µL) Master Mix(x5.5)(µL)
Milli-Q H₂O 36.5 202.4
10x Pfu Buffer with MgSO₄ 5 27.5
dNTPs 2 11
Forward VF2 Primer 2 11
Reverse VR Primer 2 11
Template DNA 2
Pfu polymerase 0.2 1.1

Added 48(µL) to each reaction.
GEL PICTURE!!!

September 14, 2010

JF

Objective: Miniprep of RFP expression construct (J04450) via Qiaquick/Qiaprep spin column. Protocol followed as per kit instructions.

---
Restriction Reactions -

Ingredient Volume(µL)

MilliQ H₂0 Water 36.1

NEBuffer 2 (10x) 5

Plasmid DNA 26.4

EcoRI-HF 1

SpeI 1

100X BSA 0.5

Ingredient Volume(µL)

MilliQ H₂0 Water 24.6

NEBuffer 2 (10x) 5

Downstream Part (dT) 17.9

XbaI 1

PstI 1

100X BSA 0.5

Ingredient Volume(µL)

MilliQ H₂0 Water 3.35

NEBuffer 2 (10x) 2.5

Destination Plasmid (pSB1C3) 17.9

EcoRI-HF .5

PstI .5

100X BSA 0.25

2(µL) was removed prior to adding enzymes for analysis on gel.
Split digestion reactions in half: dT - 2 @ 24(µL); Lum - 2 @ 24(µL); psB1C3 - 2 @ 11.5(µL)
Ran 10 minute and 60 minute reactions at 37^oC. Followed by 20 minute heat shock at 80^oC.
2(µL) was removed from the heat killed reactions for analysis on gel.

Performed 4 combinations of ligation: 10 min Restriction + 10 min Ligation; 10 min Restriction + Overnight Ligation; 60 min Restriction + 10 min Ligation; 60 min Restriction + Overnight Ligation

Ingredient 1X(µL) 2X Master Mix(x2.5)(µL)
MilliQ H₂0 Water 11 77.5
T4 Ligase Buffer (10x) 2 5
Upstream Part 2 5
Downstream Part 2 5
T4 DNA Ligase 1 2.5
Plasmid Part 2 5

Incubated 10 minute and overnight ligations at room temperature ( 25^oC). Heat killed ligase at 80^oC for 20 min.

2(µL) of each reaction was taken for analysis on gel.

Confirmed ligation with PCR analysis. Analyzed restriction, ligation and PCR on a 1.5% TAE agarose gel.

Component 1X(µL) Master Mix(x5.5)(µL)
Milli-Q H₂O 36.5 202.4
10x Pfu Buffer with MgSO₄ 5 27.5
dNTPs 2 11
Forward VF2 Primer 2 11
Reverse VR Primer 2 11
Template DNA 2
Pfu polymerase 0.2 1.1

Added 48(µL) to each reaction.
GEL PICTURE!!!

September 14, 2010

ADS

Objective: Assemble xylE-dT using 3AB assembly and 3AB assembly.

September 20, 2010

ADS

Objective: Analyze PCR of mms6 (HB) and fluorescent proteins (AV).
Method: Analyzed on 2% TAE agarose gel.
Load order:

Lane Sample Volume
Sample (µL) Volume Loading
Dye (µL)

1 HB1 10 2

2 HB2 10 2

3 HB3 10 2

4 HB4 10 2

5 HB5 10 2

6 100bp Ladder (Fermentas) 0.5 2(+10 H₂0)

7 RFP1 10 2

8 RFP2 10 2

9 RFP3 10 2

Results: ADD IMAGE

mms6 was not amplified
RFP was amplified
Expected size is ~800bp
Actual size is >1000bp
We believe that the suffix segment of the fusion primer annealed rather than the FP segment. This would cause the promoter (R0040) of J04450 to be amplified, adding ~200bp, giving a final size of >1000bp.

Objective: Confirm assembly (3 antibiotic) of K118021 and B0015 from Sept. XX, 2010 via PCR.
Method: Set up 50uL reaction mixture with 2uL of ligated DNA. Used VF2 and VR primers.
Used PFU setting on Thermocycler
Results: ADD IMAGE
Conclusion: Assembly did not work as intended.

September 21, 2010

ADS

Objective: Insert xylE (with N and C terminal fusion standards, obtained by PCR of K118021 by KG) into pSB1C3 plasmid for submission to registry.

Method:

Restriction of xylE PCR product and pSB1C3 (containing J04450 biobrick) via BioBrick Method using EcoRI-HF and PstI (Enzymes from NEB)
Ligation of xylE PCR product and pSB1C3 via BioBrick Method using T4 DNA ligase (Enzyme from NEB)
Transform into Library Efficiency Compentent DH5α Cells (Invitrogen)

Results:

Obtained too numerous to count (TNTC) colonies.
Obtained ~100 white colonies (indicating removal of RFP and insertion of new BioBrick)

Note:
Parent plasmid (from PCR) not digested, possibly K118021 moved into pSB1C3 backbone.
Follow-up:
Screen white colonies by addition of catechol to solution containing white cells.

September 21, 2010

ADS

Objective: Analyze PCR of K118021-B0015 and subsequent PCR (ADS) and PCR of <partinfo>K118021</partinfo> to add either N or C terminal fusion standard (KG).
Method: Analyze on 2% TAE agarose gel.
Load Order:

Lane Sample Volume
Sample (µL) Volume Loading
Dye (µL)

1 xylE F-S 1 5 1

2 xylE F-S 2 5 1

3 xylE F-S 3 5 1

4 xylE F-S 4 5 1

5 xylE F-S 5 5 1

6 xylE F-S 6 5 1

7 100bp Ladder (NEB) 0.5 1(+5 H₂0)

8 xylE S-F 1 5 1

9 xylE S-F 2 5 1

10 xylE S-F 3 5 1

11 xylE S-F 4 5 1

12 xylE S-F 5 5 1

13 xylE S-F 6 5 1

14 Empty

15 PCR of K118021-B0015 (ADS) 1 1

16 Empty

17 Empty

Results: ADD IMAGE

Both KG PCR (Standard-Fusion and Fusion-Standard) amplified
ADS PCR Amplified, but no insert present.

September 23, 2010

JV

Objective: Characterized catechol degradation by xylE enzyme

Method: Measured absorbance of catechol (275nm) and 2-hydroxymuconate semialdehyde (380nm).

Protocol:
1) Grow cells in M9 minimal medium
2) Take 1/10 dilution of cells
3) Introduce 1µL of 0.05M catechol solution into the cell dilution. (Final concentration of 50&microM;).
4) Quench the reaction with 5A% w/v trichloroacetate at certain time points. (0,15sec, 30sec, 45sec, 60sec, 2min, 3min, 4min, 5min, 10min).
5) Spin down cells.
6) Measure absorbance of supernatant.

Results: Cuvette used interfered with Spectra.

ADS

NOTE: In all transformations, heat shock step was missed. HOWEVER, all transformations showed significant number of colony forming units.
Objective: Move xylE (two biobrick; one with Fusion prefix, one with fusion suffix) into pSB1C3.
Method:

Restriction of xylE PCR product and pSB1C3 (containing J04450 biobrick) via BioBrick Method using EcoRI-HF and PstI (Enzymes from NEB)
Ligation of xylE PCR product and pSB1C3 via BioBrick Method using T4 DNA ligase (Enzyme from NEB)
Incubated 30 min at RT

Transform into Subcloning Efficiency Compentent DH5α Cells (Invitrogen)

Results: TBD
Follow-up: TBD

Objective: Create glycerol stocks of <partinfo>J04450</partinfo> in pSB1A3 and pSB1T3 for use in RFP-BioBrick Assembly.
Method: Transform into Subcloning Efficiency Competent DH5α Cells (Invitrogen)
Obtained all plasmid DNA from 2010 Kit Plate 1

J04450 in pSB1A3 - Well 1C
J04450 in pSB1T3 - Well 7A

Results: Obtained TNTC colonies
Follow-up:

Grow overnight cultures
Generate Glycerol Stocks
Generate Plasmid DNA via Maxiprep

Objective: Create glycerol stocks of received synthesized (Mr. Gene) signal peptides.
Method: Transform into Subcloning Efficiency Competent DH5α Cells (Invitrogen) plasmid DNA containing the following BioBricks:

1) <partinfo>K331007</partinfo> - β-lactamase Bla Signal Sequence
2) <partinfo>K331008</partinfo> - Outer Membrane Protein ompA
3) <partinfo>K331009</partinfo> - Heat Stable Toxin I
4) <partinfo>K331012</partinfo> - Penicillin Binding Protein DacA
All inserts in pMA-T vector (Standard Mr. Gene vector)

Results: Obtained TNTC Cells
Follow-up:

1) Grow overnight cultures
2) Purify pDNA
3) Move into pSB1C3 plasmid
4) Verify sequence
5) Submit to registry for sequencing

September 24, 2010

ADS

Objective: Generate plasmid DNA of <partinfo>E1010</partinfo> for downstream PCR
Method: Transform plasmid DNA into Subcloning Efficiency Competent DH5α Cells (Invitrogen)
DNA obtained from 2010 Kit Plate 1 Well 18F (E1010 in pSB2K3)
Results: Obtained TBD colonies
Follow-up:

Grow overnight cultures (Generate glycerol stocks)
Purify plasmid DNA (Generate pDNA stocks)
PCR to add terminal fusion standards

Objective: Create glycerol stocks of J04450 in pSB1K3 for use in RFP-BioBrick Assembly.
Method: Transform into Subcloning Efficiency Competent DH5α Cells (Invitrogen)
Obtained plasmid DNA from 2010 Kit Plate 1 well 5A (J04450 in pSB1K3)
Results: Obtained TNTC colonies
Follow-up:

Grow overnight cultures
Generate Glycerol Stocks
Generate Plasmid DNA via Maxiprep

September 25, 2010

JV

Objective: Extract Plasmid DNA from DH5α cells.

Method:Qiagen spin column protocol.

<partinfo>K331007</partinfo> (in pMA-T vector)
<partinfo>K331008</partinfo> (in pMA-T vector)
<partinfo>K331009</partinfo> (in pMA-T vector)
<partinfo>K331012</partinfo> (in pMA-T vector)

Cells containing plasmids were put into glycerol stocks and put into HJ's -80^oC.

Retrieved from "http://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/September"

@@ Line 290: / Line 290: @@
 ===<font color="white">JF===
 <b>Objective:</b> Miniprep of RFP expression construct (J04450) via Qiaquick/Qiaprep spin column.  Protocol followed as per kit instructions.<br>
+---
+Restriction Reactions -
+<table><table border ="3">
+<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr>
+<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>36.1</td></tr>
+<tr><td>NEBuffer 2 (10x)</td><td>5</td></tr>
+<tr><td>Plasmid DNA</td><td>26.4</td></tr>
+<tr><td>EcoRI-HF</td><td>1</td></tr>
+<tr><td>SpeI</td><td>1</td></tr>
+<tr><td>100X BSA</td><td>0.5</td></tr>
+</table>
+<table><table border ="3">
+<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr>
+<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>24.6</td></tr>
+<tr><td>NEBuffer 2 (10x)</td><td>5</td></tr>
+<tr><td>Downstream Part (dT)</td><td>17.9</td></tr>
+<tr><td>XbaI</td><td>1</td></tr>
+<tr><td>PstI</td><td>1</td></tr>
+<tr><td>100X BSA</td><td>0.5</td></tr>
+</table>
+<table><table border ="3">
+<tr><td><b>Ingredient</b></td><td>Volume(&micro;L)</td></tr>
+<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>3.35</td></tr>
+<tr><td>NEBuffer 2 (10x)</td><td>2.5</td></tr>
+<tr><td>Destination Plasmid (pSB1C3)</td><td>17.9</td></tr>
+<tr><td>EcoRI-HF</td><td>.5</td></tr>
+<tr><td>PstI</td><td>.5</td></tr>
+<tr><td>100X BSA</td><td>0.25</td></tr>
+</table>
+* 2(&micro;L) was removed prior to adding enzymes for analysis on gel.
+** Split digestion reactions in half: dT - 2 @ 24(&micro;L); Lum - 2 @ 24(&micro;L); psB1C3 - 2 @ 11.5(&micro;L)
+*** Ran 10 minute and 60 minute reactions at 37<sup>o</sup>C.  Followed by 20 minute heat shock at 80<sup>o</sup>C.
+**** 2(&micro;L) was removed from the heat killed reactions for analysis on gel.
+----
+* Performed 4 combinations of ligation: 10 min Restriction + 10 min Ligation; 10 min Restriction + Overnight Ligation; 60 min Restriction + 10 min Ligation; 60 min Restriction + Overnight Ligation
+<table><table border ="3">
+<tr><td><b>Ingredient</b><td><b>1X(&micro;L)</b><td><b>2X Master Mix(x2.5)(&micro;L)</b>
+<tr><td>MilliQ H<sub>2</sub>0 Water</td><td>11<td>77.5
+<tr><td>T4 Ligase Buffer (10x)</td><td>2<td>5
+<tr><td>Upstream Part</td><td>2<td>5
+<tr><td>Downstream Part </td><td>2<td>5
+<tr><td>T4 DNA Ligase</td><td>1<td>2.5
+<tr><td>Plasmid Part</td><td>2<td>5
+</table>
+Incubated 10 minute and overnight ligations at room temperature ( 25<sup>o</sup>C).  Heat killed ligase at 80<sup>o</sup>C for 20 min.
+* 2(&micro;L) of each reaction was taken for analysis on gel.
+----
+* Confirmed ligation with PCR analysis.  Analyzed restriction, ligation and PCR on a 1.5% TAE agarose gel.
+<table border ="3">
+<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x5.5)(&micro;L)</b>
+<tr><td>Milli-Q H<sub>2</sub>O<td>36.5<td>202.4
+<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>27.5
+<tr><td>dNTPs<td>2<td>11
+<tr><td>Forward VF2 Primer<td>2<td>11
+<tr><td>Reverse VR Primer<td>2<td>11
+<tr><td>Template DNA<td>2<td>
+<tr><td>Pfu polymerase<td>0.2<td>1.1
+</table><br>
+Added 48(&micro;L) to each reaction.
+GEL PICTURE!!!
 ==<font color="white">September 14, 2010==

Ingredient	Volume(µL)
MilliQ H₂0 Water	36.1
NEBuffer 2 (10x)	5
Upstream Part (Lumazine)	26.4
EcoRI-HF	1
SpeI	1
100X BSA	0.5

Ingredient	1X(µL)	2X Master Mix(x2.5)(µL)
MilliQ H₂0 Water	11	77.5
T4 Ligase Buffer (10x)	2	5
Upstream Part	2	5
Downstream Part	2	5
T4 DNA Ligase	1	2.5
Plasmid Part	2	5

Component	1X(µL)	Master Mix(x5.5)(µL)
Milli-Q H₂O	36.5	202.4
10x Pfu Buffer with MgSO₄	5	27.5
dNTPs	2	11
Forward VF2 Primer	2	11
Reverse VR Primer	2	11
Template DNA	2
Pfu polymerase	0.2	1.1

Lane	Sample	Volume Sample (µL)	Volume Loading Dye (µL)
1	HB1	10	2
2	HB2	10	2
3	HB3	10	2
4	HB4	10	2
5	HB5	10	2
6	100bp Ladder (Fermentas)	0.5	2(+10 H₂0)
7	RFP1	10	2
8	RFP2	10	2
9	RFP3	10	2