Team:Caltech/Week 6

From 2011.igem.org

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==July 18==
==July 18==
-
Colony PCR of positive Gibson plates from Friday<br/>
+
[[File:Caltech_7_19_Gel_1.jpg|thumb|left|Colonies 1-6 from Gibson plates; 7 and 8 are reswabs of two colonies. From left to right: DNA ladder,(PNT001; colony 1; vector), P2v, P3v, P4v, P5v, P6v, P7v, P8v, (PNT001; colony 1; insert), P3i, P4i, P5i, P6i]]
 +
[[File:Gel2_7_19_Caltech.jpg.jpg|thumb|left|from left to right: DNA ladder, (PNT001; reswab 7; insert), P8i, (16s; colony A; vector), 16Bv, 16Cv, (16s; A; insert), 16Bi, 16Ci]]Colony PCR of colonies from positive Gibson plates from Friday, for vector and insert<br/>
 +
Colony PCR of 16s plasmids, for vector and insert<br/>
Gel of short PCR parts to confirm they are being amplified<br/>
Gel of short PCR parts to confirm they are being amplified<br/>
Pulsed field gel with a larger amount of soil sample DNA<br/>
Pulsed field gel with a larger amount of soil sample DNA<br/>
Retransform pSB3K5 into Xl-10s<br/>
Retransform pSB3K5 into Xl-10s<br/>
===Results===
===Results===
 +
Gels show mostly expected single bands<br/><br/><br/><br/><br/>
==July 19==
==July 19==
-
PCR parts for Gibson assembly of pNT001, pNT003 and Joe's positive GFP control<br>
+
PCR parts for Gibson assembly of pNT001, pNT003 and Joe's positive GFP control<br/>
===Results===
===Results===
pSB3K5 plate had 0 colonies.<br/>
pSB3K5 plate had 0 colonies.<br/>
-
PFGE of soil-extracted DNA shows a smear; will do gel extraction of ~40kbp sequences for fosmid kit.
+
PFGE of soil-extracted DNA shows a smear; will do gel extraction of ~40kbp sequences for fosmid kit.<br/>
 +
 
 +
[[File:pfge7192011.jpg|thumb|lane 1 lambda mono-cut ladder, 2 blank, 3 40 kb control DNA, 4 LA River sample 9, 5 LA-River sample 10]]
Gibson PCR concentrations
Gibson PCR concentrations
Line 47: Line 52:
</tr>
</tr>
</table>
</table>
 +
 +
==July 20==
 +
Pulse field gel of soil-extracted DNA, in preparation for fosmid insertion<br/>
 +
PCR parts for Gibson assembly of pNT001, pNT003 and positive control<br/>
 +
Redo Gibson assembly of pNT001 using a smaller total mass of DNA. Gibson assemble GFP from Joe that is known to have worked before as a positive control<br/>
 +
Try transforming pSB3K5 with less DNA and as a liquid culture<br/>
 +
Prepare minimal media plates with chemical layer<br/>
 +
===Results===
 +
PCR concentrations
 +
<table border="1">
 +
<tr>
 +
<th>Part</th>
 +
<th>Concentration (ng/ul)</th>
 +
</tr>
 +
<tr>
 +
<td>R0010 for pNT003</td>
 +
<td>142.0</td>
 +
</tr>
 +
<tr>
 +
<td>K123000</td>
 +
<td>182.2</td>
 +
</tr>
 +
<tr>
 +
<td>K123003</td>
 +
<td>43.7</td>
 +
</tr>
 +
<tr>
 +
<td>promoter for GFP Gibson positive control</td>
 +
<td>74.8</td>
 +
</tr>
 +
<tr>
 +
<td>pSB4A5</td>
 +
<td>92.8</td>
 +
</tr>
 +
</table>
 +
 +
 +
 +
Minimal media plates:  BPA and DDT plates came out wrong.  Will plate DDT cultures anyway and redo.
 +
[[File:Moar_plates.png|thumb|200px|right|Minimal media plates.  Left to right:  17a-ethinylestradiol, DDT, BPA]]
 +
 +
 +
 +
==July 21==
 +
Gel extraction of 40kb LA River sample DNA from pulse field gel<br/>
 +
Transform pSB3K3 into competent cells, since the pSB3K5 transform is not working<br/>
 +
Run Gibson assembly of Joe's GFP and pNT001 and stop at different time points. Run on a gel to see what size DNA there is at different times in the reaction<br/>
 +
Try "step-wise" Gibson. Add the insert for pNT001 (3 ul) into a 10 ul reaction and incubate at 50˚C for 10 or 20 minutes before adding to a premade Gibson mix of just the vector (2 ul). Continue incubating until a total of 1 hour has passed. This may help with the self-ligation of the vector by giving the insert time to assemble if it works<br/>
 +
Plate DDT and estradiol cultures on minimal media plates
 +
===Results===
 +
[[File:7-21gibsontime.jpg|thumb|200px|lane 1 NEB 2-log ladder, 2 GFP 0 min, 3 GFP 5 min, 4 GFP 10 min, 5 GFP 15 min, 6 GFP 30 min, 7 GFP 60 min, 8 blank, 9 pNT001 0 min, 10 pNT001 5 min, 11 pNT001 10 min, 12 pNT001 15 min, 13 pNT001 30 min, 14 pNT001 60 min, 15 NEB 2-log ladder. Triangles represent increasing time (not to scale)]]
 +
Gibson Plates
 +
<table border="1">
 +
<tr>
 +
<th>Plate</th>
 +
<th>Number of Colonies</th>
 +
</tr>
 +
<tr>
 +
<td>GFP +</td>
 +
<td>10</td>
 +
</tr>
 +
<tr>
 +
<td>GFP -</td>
 +
<td>3</td>
 +
</tr>
 +
<tr>
 +
<td>pNT001 +</td>
 +
<td>29</td>
 +
</tr>
 +
<tr>
 +
<td>pNT001 -</td>
 +
<td>2</td>
 +
</tr>
 +
</table>
 +
==July 22==
 +
Gave a presentation of our project so far to Dr. Murray's bio subgroup.<br/>
 +
Sent off pNT001 and GFP Gibson colonies for sequencing.<br/>
 +
Attempted to PCR pNT001 insert from PCR'd products (part plus overlapping primers)<br/>
 +
===Results===
 +
Gibson Plates
 +
<table border="1">
 +
<tr>
 +
<th>Plate</th>
 +
<th>Number of Colonies</th>
 +
</tr>
 +
<tr>
 +
<td>pNT001 + (normal reaction)</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>pNT001 -</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>pNT001 10 minute transfer</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>pNT001 20 minute transfer</td>
 +
<td>0</td>
 +
</tr>
 +
</table>
 +
Transform Plates
 +
<table border="1">
 +
<tr>
 +
<th>Plate</th>
 +
<th>Number of Colonies</th>
 +
</tr>
 +
<tr>
 +
<td>pSB3K3</td>
 +
<td>6</td>
 +
</tr>
 +
<tr>
 +
<td>pSB3K3 (1/10 dilution)</td>
 +
<td>0</td>
 +
</tr>
 +
</table>
 +
[[File:7-22pcrpnt001.jpg|thumb|lane 1 NEB 2-log ladder, 2-6 pNT001insert]]
 +
Miniprep concentrations
 +
<table border="1">
 +
<tr>
 +
<th>Name</th>
 +
<th>concentration (ng/ul)</th>
 +
</tr>
 +
<tr>
 +
<td>GFP 1</td>
 +
<td>37.1</td>
 +
</tr>
 +
<tr>
 +
<td>GFP 2</td>
 +
<td>22.9</td>
 +
</tr>
 +
<tr>
 +
<td>pNT001 1</td>
 +
<td>29.9</td>
 +
</tr>
 +
<tr>
 +
<td>pNT001 2</td>
 +
<td>46.1</td>
 +
</tr>
 +
<tr>
 +
<td>pNT001 3</td>
 +
<td>34.5</td>
 +
</tr>
 +
<tr>
 +
<td>pNT001 4</td>
 +
<td>32.4</td>
 +
</tr>
 +
</table>
 +
PCR of the pNT001 insert was successful. The bright band around 700 bp is the correct length for the insert.<br/>
 +
==July 23==
 +
===Results===
 +
Sequencing results are back. pNT001 sample 1 was the only one with a good alignment. Blasted both GFP colonies and they contained vector only.<br/>
 +
PFGE-extracted DNA concentrated by evaporation centrifuging; nanodrop results: 9-1: 11.4ng/uL; 10-2: 29.7ng/uL<br/>
}}
}}

Latest revision as of 22:19, 26 July 2011


Caltech iGEM 2011



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References

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July 18

Colonies 1-6 from Gibson plates; 7 and 8 are reswabs of two colonies. From left to right: DNA ladder,(PNT001; colony 1; vector), P2v, P3v, P4v, P5v, P6v, P7v, P8v, (PNT001; colony 1; insert), P3i, P4i, P5i, P6i
from left to right: DNA ladder, (PNT001; reswab 7; insert), P8i, (16s; colony A; vector), 16Bv, 16Cv, (16s; A; insert), 16Bi, 16Ci
Colony PCR of colonies from positive Gibson plates from Friday, for vector and insert

Colony PCR of 16s plasmids, for vector and insert
Gel of short PCR parts to confirm they are being amplified
Pulsed field gel with a larger amount of soil sample DNA
Retransform pSB3K5 into Xl-10s

Results

Gels show mostly expected single bands




July 19

PCR parts for Gibson assembly of pNT001, pNT003 and Joe's positive GFP control

Results

pSB3K5 plate had 0 colonies.
PFGE of soil-extracted DNA shows a smear; will do gel extraction of ~40kbp sequences for fosmid kit.

lane 1 lambda mono-cut ladder, 2 blank, 3 40 kb control DNA, 4 LA River sample 9, 5 LA-River sample 10

Gibson PCR concentrations

Part Concentration (ng/ul)
R0010 for pNT001 86.4
B0014 for pNT001 128.3
B0014 for pNT003 89.1
promoter for GFP Gibson positive control 51.9
GFP for positive control 130.4
terminator for positive control 82.5

July 20

Pulse field gel of soil-extracted DNA, in preparation for fosmid insertion
PCR parts for Gibson assembly of pNT001, pNT003 and positive control
Redo Gibson assembly of pNT001 using a smaller total mass of DNA. Gibson assemble GFP from Joe that is known to have worked before as a positive control
Try transforming pSB3K5 with less DNA and as a liquid culture
Prepare minimal media plates with chemical layer

Results

PCR concentrations

Part Concentration (ng/ul)
R0010 for pNT003 142.0
K123000 182.2
K123003 43.7
promoter for GFP Gibson positive control 74.8
pSB4A5 92.8


Minimal media plates: BPA and DDT plates came out wrong. Will plate DDT cultures anyway and redo.

Minimal media plates. Left to right: 17a-ethinylestradiol, DDT, BPA


July 21

Gel extraction of 40kb LA River sample DNA from pulse field gel
Transform pSB3K3 into competent cells, since the pSB3K5 transform is not working
Run Gibson assembly of Joe's GFP and pNT001 and stop at different time points. Run on a gel to see what size DNA there is at different times in the reaction
Try "step-wise" Gibson. Add the insert for pNT001 (3 ul) into a 10 ul reaction and incubate at 50˚C for 10 or 20 minutes before adding to a premade Gibson mix of just the vector (2 ul). Continue incubating until a total of 1 hour has passed. This may help with the self-ligation of the vector by giving the insert time to assemble if it works
Plate DDT and estradiol cultures on minimal media plates

Results

lane 1 NEB 2-log ladder, 2 GFP 0 min, 3 GFP 5 min, 4 GFP 10 min, 5 GFP 15 min, 6 GFP 30 min, 7 GFP 60 min, 8 blank, 9 pNT001 0 min, 10 pNT001 5 min, 11 pNT001 10 min, 12 pNT001 15 min, 13 pNT001 30 min, 14 pNT001 60 min, 15 NEB 2-log ladder. Triangles represent increasing time (not to scale)

Gibson Plates

Plate Number of Colonies
GFP + 10
GFP - 3
pNT001 + 29
pNT001 - 2

July 22

Gave a presentation of our project so far to Dr. Murray's bio subgroup.
Sent off pNT001 and GFP Gibson colonies for sequencing.
Attempted to PCR pNT001 insert from PCR'd products (part plus overlapping primers)

Results

Gibson Plates

Plate Number of Colonies
pNT001 + (normal reaction) 0
pNT001 - 0
pNT001 10 minute transfer 0
pNT001 20 minute transfer 0

Transform Plates

Plate Number of Colonies
pSB3K3 6
pSB3K3 (1/10 dilution) 0
lane 1 NEB 2-log ladder, 2-6 pNT001insert

Miniprep concentrations

Name concentration (ng/ul)
GFP 1 37.1
GFP 2 22.9
pNT001 1 29.9
pNT001 2 46.1
pNT001 3 34.5
pNT001 4 32.4

PCR of the pNT001 insert was successful. The bright band around 700 bp is the correct length for the insert.

July 23

Results

Sequencing results are back. pNT001 sample 1 was the only one with a good alignment. Blasted both GFP colonies and they contained vector only.
PFGE-extracted DNA concentrated by evaporation centrifuging; nanodrop results: 9-1: 11.4ng/uL; 10-2: 29.7ng/uL


Retrieved from "http://2011.igem.org/Team:Caltech/Week_6"