Team:UCL/Labbook/Week16

From 2013.igem.org

(Difference between revisions)
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</p>
</p>
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<p class="body_text">
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Nanodrop readings (Tom's)
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</p>
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<p class="body_text">
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<table>
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<tr>
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<th>Sample</th>
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<th>ng/ul</th>
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<th>260/280</th>
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</tr>
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<tr>
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<td>J63A (glyc stock)</td>
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<td>62.3</td>
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<td>1.74</td>
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</tr>
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<tr>
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<td>CCB4 (4xcmp comp cells)</td>
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<td>46.8</td>
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<td>1.94</td>
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</tr>
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<tr>
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<td>CCB2 (2xcmp comp cells)</td>
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<td>57.6</td>
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<td>1.87</td>
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</tr>
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</table>
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</p>
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 +
<p class="body_text">
 +
Analytical digest of zeo+pSB1C3 (from AA1 ligation) potential clones with xba1
 +
Eight clones (AA1 col x miniprep 15/09 RC) + AA1 5xcmp mini prep were digested with xba1 following the recipe:
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</p>
 +
<p class="body_text">
 +
<table>
 +
<tr>
 +
<th>Components</th>
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<th>Volume (ul)</th>
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</tr>
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<tr>
 +
<td>DNA</td>
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<td>5</td>
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</tr>
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<tr>
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<td>Xba1</td>
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<td>1</td>
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</tr>
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<tr>
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<td>Buffer 2</td>
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<td>1</td>
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</tr>
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<tr>
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<td>BSA</td>
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<td>0.5</td>
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</tr>
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<tr>
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<td>dH2O</td>
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<td>2.5</td>
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</tr>
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<tr>
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<td>Total</td>
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<td>10</td>
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</tr>
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</table>
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</p>
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<p class="body_text">
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Samples were briefly centrifuged and incubated at 37O C for circa 3 hours. After that, samples were supplemented with 3 ul dye and run on a gel.
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</p>
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<p class="body_text">
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<u>Purification of 8 PCR reactions to amplify Zeo and BB hangers</u>
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</p>
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<p class="body_text">
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These reactions (total volume of 400 ul) were left in the thermocycler at 4O C overnight. The samples were run on a gel together with 70 ul dye and the correct bands (1.8 kb) were gel extracted. This gel was purified and eluted in 40 ul Elution Buffer. The nanodrop readings of these were of 91.9 ul/ul with purity (260/280) of 1.96.
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</p>
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<p class="body_text">
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<u>Preparative digest of amplified zeocin using EcoR1 and Pst1</u>
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</p>
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<p class="body_text">
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<table>
 +
<tr>
 +
<th>Components</th>
 +
<th>Volume (ul)</th>
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</tr>
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<tr>
 +
<td>DNA</td>
 +
<td>35</td>
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</tr>
 +
<tr>
 +
<td>EcoR1</td>
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<td>7</td>
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</tr>
 +
<tr>
 +
<td>Pst1</td>
 +
<td>7</td>
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</tr>
 +
<tr>
 +
<td>Buffer 2</td>
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<td>10</td>
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</tr>
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<tr>
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<td>BSA</td>
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<td>4</td>
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</tr>
 +
<tr>
 +
<td>dH2O</td>
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<td>37</td>
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</tr>
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<tr>
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<td>Total</td>
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<td>100</td>
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</tr>
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</table>
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</p>
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 +
<p class="body_text">
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These reactions were Incubated for 2 hours at 37OC.
 +
After, the digest was purified with PCR purification kit. Nanodrop result was found to be 60.4 ng/ul (260/280=1.82, the purity).This was taken further for a ligation as per the following recipe:
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</p>
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<p class="body_text">
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<u>Ligation 5 for zeocin and pSB1C3</u>
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</p>
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<p class="body_text">
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pSB1C3 concentration = 50 ng/ul
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</p>
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<p class="body_text">
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Zeocin insert concentration = 25 ng/ul
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</p>
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<p class="body_text">
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<table>
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<tr>
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<th>Component</th>
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<th>Lig 1 (ul)</th>
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<th>Lig 2 (ul)</th>
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<th>Lig 3 (ul) control</th>
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<th>Lig 4 (ul) control</th>
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</tr>
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<tr>
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<td>pSB1C3</td>
 +
<td>2</td>
 +
<td>2</td>
 +
<td>2</td>
 +
<td>0</td>
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</tr>
 +
<tr>
 +
<td>Zeo</td>
 +
<td>2</td>
 +
<td>2.5</td>
 +
<td>0</td>
 +
<td>2</td>
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</tr>
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<tr>
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<td>Quick T4 ligase</td>
 +
<td>1</td>
 +
<td>1</td>
 +
<td>1</td>
 +
<td>1</td>
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</tr>
 +
<tr>
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<td>T4 ligase buffer</td>
 +
<td>10</td>
 +
<td>10</td>
 +
<td>10</td>
 +
<td>10</td>
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</tr>
 +
<tr>
 +
<td>dH2O</td>
 +
<td>5</td>
 +
<td>4.5</td>
 +
<td>7</td>
 +
<td>7</td>
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</tr>
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<tr>
 +
<td>Total</td>
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<td>20</td>
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<td>20</td>
 +
<td>20</td>
 +
<td>20</td>
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</tr>
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</table>
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</p>
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 +
<p class="body_text">
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These tubes were incubated at room temperature for 5 minutes then used 5 ul from each for transformation using W3110 cells. Plated 10 ul, 90 ul on 5xcmp selective plates and then incubated overnight at 37OC. Next day, there was no growth on neither plates
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</p>
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<p class="body_text">
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Two gel were loaded using xba1 digests of recombinant zeo candidats
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</p>
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<p class="body_text">
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1.Ladder, 1-9 Xba1 candidate digest, 25 D, 26U, 26D, 26U, 28D, 28U.
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2. Ladder, 1D, 1U, 3D, 3U, 12D, 12U, 13D, 13U, 15D, 15U, 21D, 21U, J63A D, J63A U, AuxD, AuxU
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<b>INSERT GEL PHOTO HERE</b>
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</p>
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<p class="body_text">
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Ten ul of sample 1(showed correct band pattern) was used from glycerol stock to make an inoculation in 4xcmp 10 ml LB broth.
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</p.
</div>
</div>
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<!-- END CONTENT ------------------------------------------------------------------------------------------------------>
<!-- END CONTENT ------------------------------------------------------------------------------------------------------>

Revision as of 09:05, 4 October 2013

Lab Weeks

Week 16

Bacterial Labs

Results of the inoculations of transformations of 13/09 in 4xcmp: all showed growth apart from falcons 5.3, 5.2, 1.14 and 1.2. Glycerol stocks of the rest of 19 inoculations were made.

Minipreps of the above inoculations were made only for 1.3, 1.7, 1.9, 1.10, 1.11, 1.15, 1.20 and 5.1 due to lack of chromatographic columns.

Nanodrop result of the above minipreps showed concentration values below 16.0 ng/ul. Analytical digest of miniprep samples 1-9 (prepared the day before) as well as of 5.1 and 1.15 (which showed concentrations of about 15 ng/ul) with E and P

Components Volume (ul)
DNA 5
EcoR1 1
Pst1 1
Buffer 3 1
BSA 0.5
dH2O 1.5
Total 10

INSERT IMAGE OF GEL 16/09 ZEO BB CANDIDATES DIGESTED WITH E&P FROM MINIPREPS OF COLONIES TRANSFORMED ON 13/09

Prep digest of miniprep of J632014 with E and P in order to keep up the pSB1C3 stocks

Components Cut Uncut Control - pSecTag2A
J632014 DNA 25 5 10
EocR1 2 0 0
Pst1 2 0 0
Buffer 3 4 0 0
BSA 0.5 0 0
dH2O 6.5 5 0
Total 40 10 10

These were incubated for 2 hours at 37O C. Casey ran a gel of the following samples in the next order: Zeo BB: 26D, 26u; 28D, 28u; AuxD, AuxU INSERT IMAGE OF GEL HERE PLS

Gel1: E+P double digest and P, single digest (Auxin bb): 3, 12, 13, 15. Gel2: D digest 26, 28 and Auxin bb. Minipreps: 26, 28, 3, 12, 13, 15, 21 (sample of which DNA is waiting to be eluted)

Tuesday 17th September

Miniprep of the 16 tubes of inoculations of MMP9 as well as tube 21 of which DNA had to be eluted.

Nanodrop results of the minipreps

Tube no. ng/ul 260/280
1 20.6 1.9
2 26.9 1.64
4 22.0 3.2
5 4.8 2.16
6 22.7 2.99
7 16.4 2.41
8 16.9 2.01
9 7.1 1.49
10 27.3 1.79
11 16.0 1.72
12 22.2 1.41
13 11.0 1.73
14 12.0 1.6
15 18.4 1.57
17 45.1 1.62
18 9.2 1.72
21 (kc) 21.3 1.81

Nanodrop readings (Tom's)

Sample ng/ul 260/280
J63A (glyc stock) 62.3 1.74
CCB4 (4xcmp comp cells) 46.8 1.94
CCB2 (2xcmp comp cells) 57.6 1.87

Analytical digest of zeo+pSB1C3 (from AA1 ligation) potential clones with xba1 Eight clones (AA1 col x miniprep 15/09 RC) + AA1 5xcmp mini prep were digested with xba1 following the recipe:

Components Volume (ul)
DNA 5
Xba1 1
Buffer 2 1
BSA 0.5
dH2O 2.5
Total 10

Samples were briefly centrifuged and incubated at 37O C for circa 3 hours. After that, samples were supplemented with 3 ul dye and run on a gel.

Purification of 8 PCR reactions to amplify Zeo and BB hangers

These reactions (total volume of 400 ul) were left in the thermocycler at 4O C overnight. The samples were run on a gel together with 70 ul dye and the correct bands (1.8 kb) were gel extracted. This gel was purified and eluted in 40 ul Elution Buffer. The nanodrop readings of these were of 91.9 ul/ul with purity (260/280) of 1.96.

Preparative digest of amplified zeocin using EcoR1 and Pst1

Components Volume (ul)
DNA 35
EcoR1 7
Pst1 7
Buffer 2 10
BSA 4
dH2O 37
Total 100

These reactions were Incubated for 2 hours at 37OC. After, the digest was purified with PCR purification kit. Nanodrop result was found to be 60.4 ng/ul (260/280=1.82, the purity).This was taken further for a ligation as per the following recipe:

Ligation 5 for zeocin and pSB1C3

pSB1C3 concentration = 50 ng/ul

Zeocin insert concentration = 25 ng/ul

Component Lig 1 (ul) Lig 2 (ul) Lig 3 (ul) control Lig 4 (ul) control
pSB1C3 2 2 2 0
Zeo 2 2.5 0 2
Quick T4 ligase 1 1 1 1
T4 ligase buffer 10 10 10 10
dH2O 5 4.5 7 7
Total 20 20 20 20

These tubes were incubated at room temperature for 5 minutes then used 5 ul from each for transformation using W3110 cells. Plated 10 ul, 90 ul on 5xcmp selective plates and then incubated overnight at 37OC. Next day, there was no growth on neither plates

Two gel were loaded using xba1 digests of recombinant zeo candidats

1.Ladder, 1-9 Xba1 candidate digest, 25 D, 26U, 26D, 26U, 28D, 28U. 2. Ladder, 1D, 1U, 3D, 3U, 12D, 12U, 13D, 13U, 15D, 15U, 21D, 21U, J63A D, J63A U, AuxD, AuxU INSERT GEL PHOTO HERE

Ten ul of sample 1(showed correct band pattern) was used from glycerol stock to make an inoculation in 4xcmp 10 ml LB broth.