User:University of Washington/8 July 2008

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·Testing DH5α and S17-1+RP4 resistance to Chloremphenicol and Ampicillin.<br\>
·Testing DH5α and S17-1+RP4 resistance to Chloremphenicol and Ampicillin.<br\>
-
·Testing S17-1 to DH5α conjugation using RP4 plasmid.
+
·Testing S17-1 to DH5α conjugation using RP4 plasmid and [[Protocols| conjugation protocol #1]].
-
·Make glycerol stock of S17-1+RP4
+
·Make [[UW Glycerol Stocks| glycerol stock]] of S17-1+RP4
-
[[UW Glycerol Stocks]]
+
== Non-RP4 Conjugation ==
== Non-RP4 Conjugation ==
·Plate out CV13(Yep13) and pDPT51
·Plate out CV13(Yep13) and pDPT51
 +
 +
== LuxR from pLac ==
 +
 +
- Made glycerol stocks of R0010 and I763004.
 +
 +
- Miniprepped and sent in for sequencing part I763004.
 +
 +
== LuxR from AraC and TetR ==
 +
 +
- Cells with AraC plasmid grew on Amp plate and was stored in the fridge. (Note: The cells were spread by glass rod instead of scraping using the metal rod, so single colonies can be found only on the side of the plate)
 +
 +
- Miniprepped 5 cultures of AraC.
 +
 +
- Ran gel (5 ul DNA + 2 ul dye) and found that five plasmid from 5 tubes have the same length. The plasmid was then combined into one tube.
 +
 +
- Nanodropped AraC plasmid: 201.6 ng/ml; 260/280 = 1.89; 260/230 = 2.17
 +
 +
- Performed the first part of QuikChange Mutagenesis
 +
*Dilute Primers(100 pmole/ul) 1:10 ==> 10pmole/ul with deionized water.
 +
*Mix(2 reactions in 2 PRC tubes))
 +
<table>
 +
<tr>
 +
<th>Materials</th><th>Reaction#1</th><th>Reaction#2</th>
 +
</tr>
 +
<tr>
 +
<td>AraC plasmid</td><td>39.3 ul</td><td>39.5 ul</td>
 +
</tr>
 +
<tr>
 +
<td>10X pfuTurbo reaction buffer</td><td>5 ul</td><td>5 ul</td>
 +
</tr>
 +
<tr>
 +
<td>dNTP mix</td><td>1 ul</td><td>1 ul</td>
 +
</tr>
 +
<tr>
 +
<td>primer AraC-F</td><td>0.806 ul AraC1F</td><td>0.728 ul AraC2F</td>
 +
</tr>
 +
<tr>
 +
<td>primer AraC-R</td><td>0.806 ul AraC1R</td><td>0.728 ul AraC2R</td>
 +
</tr>
 +
<tr>
 +
<td>DMSO</td><td>3 ul</td><td>3 ul</td>
 +
</tr>
 +
</table>
 +
*add 1 ul pfuTurbo in both
 +
*Temperature Cycle(There are small changes from our [[Team:University_of_Washington/Protocols|protocol page]].
 +
<table>
 +
<tr>
 +
  <th>Segment</th><th>Cycles</th><th>Temperature</th><th>Time</th>
 +
</tr>
 +
<tr>
 +
  <td>1</td><td>1</td><td>95°C</td><td><font color="red">2 minute</font></td>
 +
</tr>
 +
<tr>
 +
  <td rowspan=3>2</td><td rowspan=3>18</td><td>95°C</td><td>50 seconds</td>
 +
</tr>
 +
<tr>
 +
  <td>60°C</td><td>50 seconds</td>
 +
</tr>
 +
<tr>
 +
  <td>68°C</td><td><font color="red">3 mins</font></td>
 +
</tr>
 +
<tr>
 +
  <td>3</td><td>1</td><td>68°C</td><td>7 minutes</td>
 +
</tr>
 +
<tr>
 +
  <td rowspan="3">let it sit in 4°C overnight</td>
 +
</tr>
 +
</table>
 +
 +
== Lambda Red Recombineering of RP4 (Bryan) ==
 +
 +
Ordered pKD76 helper plasmid from Yale CGSC, containing Lambda Red recombinatory genes and a CamR cassette.
 +
 +
Contacted Don Court at Center for Cancer Research for Lambda Red recombineering advice.  Received protocols.
 +
 +
----
----
Back to [[Team:University_of_Washington/Notebook#Notebook]]
Back to [[Team:University_of_Washington/Notebook#Notebook]]

Latest revision as of 00:58, 12 July 2008

Contents

BioBrick Promoter Construct Sequencing

- Because the previous forward sequencing reaction and analysis yielded high-noise results, the forward sequencing protocol was carried out a second time.

- VF2 primer (100pmol/uL) was diluted 1 to 100 with distilled water.

- 8 uL of diluted VF2 primer (1pmol/uL) was added to four Eppendorf tubes.

- 4 uL of miniprepped plasmid DNA for parts I20260, I20268, I20269, and I20270 were added to tubes of primer, after appropriate labeling of the tubes.

- Plasmid template and VF2 primer solutions were resubmitted to the UW DNA Sequencing Facility for reaction and analysis.

- The index and middle fingers were crossed.

RP4 Conjugation

·Testing DH5α and S17-1+RP4 resistance to Chloremphenicol and Ampicillin.
·Testing S17-1 to DH5α conjugation using RP4 plasmid and conjugation protocol #1.

·Make glycerol stock of S17-1+RP4

Non-RP4 Conjugation

·Plate out CV13(Yep13) and pDPT51

LuxR from pLac

- Made glycerol stocks of R0010 and I763004.

- Miniprepped and sent in for sequencing part I763004.

LuxR from AraC and TetR

- Cells with AraC plasmid grew on Amp plate and was stored in the fridge. (Note: The cells were spread by glass rod instead of scraping using the metal rod, so single colonies can be found only on the side of the plate)

- Miniprepped 5 cultures of AraC.

- Ran gel (5 ul DNA + 2 ul dye) and found that five plasmid from 5 tubes have the same length. The plasmid was then combined into one tube.

- Nanodropped AraC plasmid: 201.6 ng/ml; 260/280 = 1.89; 260/230 = 2.17

- Performed the first part of QuikChange Mutagenesis

  • Dilute Primers(100 pmole/ul) 1:10 ==> 10pmole/ul with deionized water.
  • Mix(2 reactions in 2 PRC tubes))
MaterialsReaction#1Reaction#2
AraC plasmid39.3 ul39.5 ul
10X pfuTurbo reaction buffer5 ul5 ul
dNTP mix1 ul1 ul
primer AraC-F0.806 ul AraC1F0.728 ul AraC2F
primer AraC-R0.806 ul AraC1R0.728 ul AraC2R
DMSO3 ul3 ul
  • add 1 ul pfuTurbo in both
  • Temperature Cycle(There are small changes from our protocol page.
SegmentCyclesTemperatureTime
1195°C2 minute
21895°C50 seconds
60°C50 seconds
68°C3 mins
3168°C7 minutes
let it sit in 4°C overnight

Lambda Red Recombineering of RP4 (Bryan)

Ordered pKD76 helper plasmid from Yale CGSC, containing Lambda Red recombinatory genes and a CamR cassette.

Contacted Don Court at Center for Cancer Research for Lambda Red recombineering advice. Received protocols.


Back to Team:University_of_Washington/Notebook#Notebook

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