Team:Caltech/Week 5

From 2011.igem.org

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==July 12==
==July 12==
[[File:7-12pcrgel.jpg|thumb|from left to right: 1 ladder, 2 R0010, 3 K123000, 4 B0014 for pNT002, 5 R0040, 6 K123001, 7 B0014 for pNT003, 8 B0014 for pNT001]]
[[File:7-12pcrgel.jpg|thumb|from left to right: 1 ladder, 2 R0010, 3 K123000, 4 B0014 for pNT002, 5 R0040, 6 K123001, 7 B0014 for pNT003, 8 B0014 for pNT001]]
-
Transformed competent cells with pSB3C5.
+
Transformed competent cells with pSB3C5.<br/>
-
Ran PCR of parts for pNT001 and pNT002 Gibson assembly.
+
Ran PCR of parts for pNT001 and pNT002 Gibson assembly.<br/>
-
Started pulsed field gel electrophoresis of some of our LA River DNA obtained by the Mo Bio kits in week 2.
+
Started pulsed field gel electrophoresis of some of our LA River DNA obtained by the Mo Bio kits in week 2.<br/>
===Results===
===Results===
All of the parts for pNT001, pNT002 and B0014 were PCR'd with primers for Gibson assembly. All lanes except for lanes 4 and 5 had single bands, indicating that the parts were successfully amplified.
All of the parts for pNT001, pNT002 and B0014 were PCR'd with primers for Gibson assembly. All lanes except for lanes 4 and 5 had single bands, indicating that the parts were successfully amplified.
==July 13==
==July 13==
-
Retrieved and imaged pulsed field gel.<br>
+
Retrieved and imaged pulsed field gel.<br/>
-
Ran PCR of R0040 and B0014 with primers that failed yesterday.<br>
+
Ran PCR of R0040 and B0014 with primers that failed yesterday.<br/>
-
Purified PCR products for Gibson of pNT001 and pNT003.<br>
+
Purified PCR products for Gibson of pNT001 and pNT003.<br/>
-
Started overnights of pSB3C5.<br>
+
Started overnights of pSB3C5.<br/>
-
Replated some pSB3C5<br>
+
Replated some pSB3C5<br/>
===Results===
===Results===
<p>The pulsed field gel had to be restained after running. The ladder and control appeared, but none of our LA River sample DNA. Since the ladder ranges from 5kb to 50 kb, we should be able to see some smears in the experimental lanes, as we ran a short gel last week and found that the sample DNA was greater than 10kb. Since the ladder does not seem well separated, we will try running the gel again with different parameters.</p>
<p>The pulsed field gel had to be restained after running. The ladder and control appeared, but none of our LA River sample DNA. Since the ladder ranges from 5kb to 50 kb, we should be able to see some smears in the experimental lanes, as we ran a short gel last week and found that the sample DNA was greater than 10kb. Since the ladder does not seem well separated, we will try running the gel again with different parameters.</p>
-
The pSB3C5 had many colonies, but none of them are red. This is strange because the plasmid contains BBa_J04450 as an insert, a RFP. We are replating on a new chlor plate and starting overnights to send off for sequencing to make sure the plasmid is okay.<br>
+
The pSB3C5 had many colonies, but none of them are red. This is strange because the plasmid contains BBa_J04450 as an insert, a RFP. We are replating on a new chlor plate and starting overnights to send off for sequencing to make sure the plasmid is okay.<br/>
-
We redid the PCR of R0040 and B0014, but no bands appeared in the gel.<br>
+
We redid the PCR of R0040 and B0014, but no bands appeared in the gel.<br/>
-
[[File:7-13pulsedgel.jpg|thumb|lanes 1 lambda mono cut ladder; 2 blank; 3 fosmid kit control; 4 LA River location 9; LA River location 10]]
+
[[File:7-13pulsedgel.jpg|thumb|lanes 1 lambda mono cut ladder; 2 blank; 1 fosmid kit control (100 ng); 9-1 Part(2000 ng); 10-1 Part(300 ng); 4 LA River location 9; LA River location 10]]
PCR Purifications for Gibson
PCR Purifications for Gibson
<table border="1">
<table border="1">
Line 70: Line 70:
==July 14==
==July 14==
 +
<p>Miniprep overnights and send off for sequencing<br/>
 +
Begin 16S sequencing by PCRing the samples with 16s universal primers and the vector we are using<br/>
 +
Try to PCR the miniprep DNA with the pSB3C5 primers<br/>
 +
Do Gibson with the PCR parts that worked</p>
 +
 +
'''Labels for 16s sequencing PCR:'''
 +
* 9-1 (1/10): 9a
 +
* 9-1 (1/100): 9b
 +
* 9-1 (1/1000): 9c
 +
* 10-3 (1/10): 10a
 +
* 10-3 (1/100): 10b
 +
* 10-3 (1/1000): 10c
 +
* Control 1: C1
 +
* Control 2: C2
 +
 +
'''Labels for PCR Miniprep:'''
 +
* pSB3C5 (1/100)- HA
 +
* pSB3C5 (1/100)- HB
 +
* pSB3C5 (1/100)- HC
 +
* pSB3C5 (1/1000)- TA
 +
* pSB3C5 (1/1000)- TB
 +
* pSB3C5 (1/1000)- TC
===Results===
===Results===
Line 91: Line 113:
</tr>
</tr>
</table>
</table>
 +
<gallery>
 +
File:Gel 2 7-14.JPG|lane 1 ladder; 2 sample 9; 3 sample 9; 4 sample 9; 5 sample 10; 6 sample 10; 7 sample 10; 8 negative control
 +
File:Gel 3 7-14.JPG|lane 1 ladder; 2-7 pSB3C5 minipreps, differing dilutions
 +
File:Gel 4 7-14.JPG|ane 1 ladder; 2-4 pET53EST (V1, V2, V3)
 +
</gallery>
 +
==July 15==
 +
Run gel of LA River samples. Perhaps the freezing and thawing has degraded the DNA further.<br/>
 +
Redo PCR of B0014 and R0040 with different annealing temperatures.<br/>
 +
Redo Gibson of pNT001 and pNT003, doing 10 ul reactions, transform into Joe's competent cells<br/>
 +
Make kanamycin stock and plates.<br/>
 +
Gibson assemble 16s with the vector<br/>
 +
Transform cells with pSB3K5, because we can't use pSB3C5 in XL-10 Golds<br/>
 +
===Results===
 +
Gibson Plates
 +
<table border="1">
 +
<tr>
 +
<th>Plate</th>
 +
<th>Number of Colonies</th>
 +
</tr>
 +
<tr>
 +
<td>pNT001 +</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>pNT001 -</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>pNT003 +</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>pNT003 -</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>vector (pSB4A5 only)</td>
 +
<td>0</td>
 +
</tr>
 +
</table>
 +
Perhaps our cells are not being very competent again. We should do a 20 ul reaction instead of a 10.5 ul reaction so we can retransform if necessary. Also, we should change amounts of DNA in the reaction. We've been diluting the DNA and trying to get all the parts within a 10 ng range. <br/>
 +
[[File:7-15larivergel.jpg|thumb|Lane 1 ladder, 2-4 sample 9, 5-7 sample 10]]
 +
The LA River DNA has been degraded to be a smear between .1-10kb, too short to use in the pulsed field gel and the fosmid kit.
 +
==July 16==
 +
===Results===
 +
<p>We are getting a lot of self-ligation of the vector.<br/>
 +
Joe's cells are more competent than ours. We used our XL-10 Golds to transform pSB3K5 and had 0 colonies in the plate. We will need to redo the transformation.</p><br/>
 +
Gibson Plates
 +
<table border="1">
 +
<tr>
 +
<th>Plate</th>
 +
<th>Number of Colonies</th>
 +
</tr>
 +
<tr>
 +
<td>pNT001 +</td>
 +
<td>6</td>
 +
</tr>
 +
<tr>
 +
<td>pNT001 -</td>
 +
<td>8</td>
 +
</tr>
 +
<tr>
 +
<td>pNT003 +</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>pNT003 -</td>
 +
<td>12</td>
 +
</tr>
 +
<tr>
 +
<td>vector (pSB4A5 only)</td>
 +
<td>76</td>
 +
</tr>
 +
<tr>
 +
<td>16s sample 9a</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>16s sample 9b</td>
 +
<td>0</td>
 +
</tr>
 +
<tr>
 +
<td>16s sample 10a</td>
 +
<td>1</td>
 +
</tr>
 +
<tr>
 +
<td>16s sample 10b</td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td>16s vector only (pET53DEST)</td>
 +
<td>1</td>
 +
</tr></table>
}}
}}

Latest revision as of 18:25, 20 July 2011


Caltech iGEM 2011



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July 11

1 ladder, 2 R0010, 3 K123003, 4 B0014, 5 pSB4A5

Miniprep pSB4A5
Run PCR to amplify [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_K123003 K123003], [http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_PSB4A5 PSB4A5]
Run gel of PCR products
Redo PCR of [http://partsregistry.org/Part:BBa_B0014 B0014]

Results

The PCR of parts for pNT003 was successful except for the terminator (B0014), as there is no band in that lane.

July 12

from left to right: 1 ladder, 2 R0010, 3 K123000, 4 B0014 for pNT002, 5 R0040, 6 K123001, 7 B0014 for pNT003, 8 B0014 for pNT001

Transformed competent cells with pSB3C5.
Ran PCR of parts for pNT001 and pNT002 Gibson assembly.
Started pulsed field gel electrophoresis of some of our LA River DNA obtained by the Mo Bio kits in week 2.

Results

All of the parts for pNT001, pNT002 and B0014 were PCR'd with primers for Gibson assembly. All lanes except for lanes 4 and 5 had single bands, indicating that the parts were successfully amplified.

July 13

Retrieved and imaged pulsed field gel.
Ran PCR of R0040 and B0014 with primers that failed yesterday.
Purified PCR products for Gibson of pNT001 and pNT003.
Started overnights of pSB3C5.
Replated some pSB3C5

Results

The pulsed field gel had to be restained after running. The ladder and control appeared, but none of our LA River sample DNA. Since the ladder ranges from 5kb to 50 kb, we should be able to see some smears in the experimental lanes, as we ran a short gel last week and found that the sample DNA was greater than 10kb. Since the ladder does not seem well separated, we will try running the gel again with different parameters.

The pSB3C5 had many colonies, but none of them are red. This is strange because the plasmid contains BBa_J04450 as an insert, a RFP. We are replating on a new chlor plate and starting overnights to send off for sequencing to make sure the plasmid is okay.
We redid the PCR of R0040 and B0014, but no bands appeared in the gel.

lanes 1 lambda mono cut ladder; 2 blank; 1 fosmid kit control (100 ng); 9-1 Part(2000 ng); 10-1 Part(300 ng); 4 LA River location 9; LA River location 10

PCR Purifications for Gibson

Part Concentration (ng/ul)
R0010 for pNT003 130.5
K123003 99.9
pSB4A5 + prefix and suffix 90.4
R0010 for pNT001 142.5
K123000 111.4
K123001 123.0
B0014 for pNT003 119.9
B0014 for pNT001 139.7

July 14

Miniprep overnights and send off for sequencing
Begin 16S sequencing by PCRing the samples with 16s universal primers and the vector we are using
Try to PCR the miniprep DNA with the pSB3C5 primers
Do Gibson with the PCR parts that worked

Labels for 16s sequencing PCR:

  • 9-1 (1/10): 9a
  • 9-1 (1/100): 9b
  • 9-1 (1/1000): 9c
  • 10-3 (1/10): 10a
  • 10-3 (1/100): 10b
  • 10-3 (1/1000): 10c
  • Control 1: C1
  • Control 2: C2

Labels for PCR Miniprep:

  • pSB3C5 (1/100)- HA
  • pSB3C5 (1/100)- HB
  • pSB3C5 (1/100)- HC
  • pSB3C5 (1/1000)- TA
  • pSB3C5 (1/1000)- TB
  • pSB3C5 (1/1000)- TC

Results

The minipreps of the overnight cultures all had very low concentrations of DNA.

Miniprep Concentration (ng/ul)
PSB3C5 A 3.1
PSB3C5 B 4.0
PSB3C5 C 3.6

lane 1 ladder; 2 sample 9; 3 sample 9; 4 sample 9; 5 sample 10; 6 sample 10; 7 sample 10; 8 negative control

lane 1 ladder; 2-7 pSB3C5 minipreps, differing dilutions

ane 1 ladder; 2-4 pET53EST (V1, V2, V3)

July 15

Run gel of LA River samples. Perhaps the freezing and thawing has degraded the DNA further.
Redo PCR of B0014 and R0040 with different annealing temperatures.
Redo Gibson of pNT001 and pNT003, doing 10 ul reactions, transform into Joe's competent cells
Make kanamycin stock and plates.
Gibson assemble 16s with the vector
Transform cells with pSB3K5, because we can't use pSB3C5 in XL-10 Golds

Results

Gibson Plates

Plate Number of Colonies
pNT001 + 0
pNT001 - 0
pNT003 + 0
pNT003 - 0
vector (pSB4A5 only) 0

Perhaps our cells are not being very competent again. We should do a 20 ul reaction instead of a 10.5 ul reaction so we can retransform if necessary. Also, we should change amounts of DNA in the reaction. We've been diluting the DNA and trying to get all the parts within a 10 ng range.

Lane 1 ladder, 2-4 sample 9, 5-7 sample 10

The LA River DNA has been degraded to be a smear between .1-10kb, too short to use in the pulsed field gel and the fosmid kit.

July 16

Results

We are getting a lot of self-ligation of the vector.
Joe's cells are more competent than ours. We used our XL-10 Golds to transform pSB3K5 and had 0 colonies in the plate. We will need to redo the transformation.


Gibson Plates

Plate Number of Colonies
pNT001 + 6
pNT001 - 8
pNT003 + 0
pNT003 - 12
vector (pSB4A5 only) 76
16s sample 9a 0
16s sample 9b 0
16s sample 10a 1
16s sample 10b 2
16s vector only (pET53DEST) 1


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