Team:Groningen/Labwork/1 August 2013

From 2013.igem.org

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<h2>Mirjam</h2>
<h2>Mirjam</h2>
Plates for the transformation of &Delta;Des, &Delta;CheY and &Delta;Des/&Delta;CheY did not show any colonies. The plates for eYFP showed some colonies on the plates with the transformation to <i>E.coli</i>. The plates with RFP and the ligation of Pdes/Chey did not show any colonies. Also the plates with a transformation of GFP240 and GFP0840 showed colonies.
Plates for the transformation of &Delta;Des, &Delta;CheY and &Delta;Des/&Delta;CheY did not show any colonies. The plates for eYFP showed some colonies on the plates with the transformation to <i>E.coli</i>. The plates with RFP and the ligation of Pdes/Chey did not show any colonies. Also the plates with a transformation of GFP240 and GFP0840 showed colonies.
-
<br>Inoculated the colonies into LB with ampicillin to perform a miniprep later on.
+
<br>Inoculated the colonies into LB with ampicillin to perform a miniprep later on. Miniprep analysis is done and the products are used to perform a restriction digestion. This showed that one of the eYFP colonies is a correct transformant. This transformant is plated on LB agar plates with IPTG and Amp resistance.
-
<br>Made a new restriction digestion for RFP, eYFP and BBa_k823823 + LacI with EcoRI and PstI. Did a gel purification to obtain the purified fragment.
+
<br>Made a new restriction digestion for RFP, eYFP and BBa_k823823 + LacI with EcoRI and PstI.  
<br>Inoculated <i>B.subtilis</i> colonies for transformation.
<br>Inoculated <i>B.subtilis</i> colonies for transformation.
<br>Made a restriction for Pdes with EcoRI and BamHI, for CheY with BamHI and PstI and for BBa_k823823 with EcoRI and PstI.
<br>Made a restriction for Pdes with EcoRI and BamHI, for CheY with BamHI and PstI and for BBa_k823823 with EcoRI and PstI.
 +
<br>Heat inactivated the restriction digestions of Des Up, Tet, Des down, CheY up, spec, CheY down and Pdes.
 +
<br>Did a PCR clean up for CheY and BBa_k823823.
 +
<br>Did a ligation reaction for Des Up, Tet and Des down as well as a ligation reaction for CheY up, spec and CheY down. Heat inactivated these reactions and did a transformation to <i>B.subtilis</i>.
 +
<br>Made a ligation reaction for Pdes and CheY and heat inactivated the reaction. This ligation reaction is ligated into BBa_k823823. Also this ligation is heat inactivated and the reaction is transformed to <i>E.coli</i>.
 +
<br>Ligation reactions RFP and eYFP with BBa_k823823 + LacI are made and heat inactivated. Then they are transformed into <i>E. coli</i>.
 +
<br>Because the <i>B.subtilis</i> culture didn't grow that well, it is decided to plate a fresh stock on agar. If todays reaction fails. There is another chance tomorrow.
 +
<p>
<p>
<h2>Chaline</h2>
<h2>Chaline</h2>
 +
Made a miniprep of eYFP and RFP to get a higher concentration.
 +
<br>Did a gel purification to obtain purified fragments of eYFP, RFP and backbone BBa_k823823 + LacI.
<p>
<p>
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The plates prepared yesterday show colonies. 4 colonies per plate are inoculated in LB + Ampicillin in order to isolate the plasmid on the day after and check for the presence of the desired insert.
The plates prepared yesterday show colonies. 4 colonies per plate are inoculated in LB + Ampicillin in order to isolate the plasmid on the day after and check for the presence of the desired insert.
<br>
<br>
-
<br>To the tube, containing the purified BBa_K823023 + lacI digested with XbaI and PstI, 1&micro;l SpeI is added and the tube is incubated for 1h at 37&deg;C. After the treatment the backbone is ligated with BBa_E0840 and BBa_E0240 (already digested and purified yesterday with XbaI and PstI). The two ligation products are transformed into <i>E. Coli</i> DH5&alpha; and plated on LB + Ampicillin.
+
<br>To the tube, containing the purified BBa_K823023 + lacI digested with XbaI and PstI, 1&micro;l SpeI is added and the tube is incubated for 1h at 37&deg;C. After the treatment the backbone is ligated (3:1 insert:vector ratio) with BBa_E0840 and BBa_E0240 (already digested and purified yesterday with XbaI and PstI). The two ligation products are transformed into <i>E. Coli</i> DH5&alpha; and plated on LB + Ampicillin.
<br>
<br>
<br><i>Bacillus Subtilis</i> is inoculated in LB.
<br><i>Bacillus Subtilis</i> is inoculated in LB.
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<table border = "1">
<table border = "1">
<tr>
<tr>
-
<dr>Sample</dr>
+
<td>Sample</td>
-
<dr>Strain</dr>
+
<td>Strain</td>
-
<dr>Selection marker</dr>
+
<td>Selection marker</td>
</tr>
</tr>
<tr>
<tr>
-
<dr>1</dr>
+
<td>1</td>
-
<dr>Delta cheY 1</dr>
+
<td>Delta cheY 1</td>
-
<dr>5 ug/ml cm</dr>
+
<td>5 ug/ml cm</td>
</tr>
</tr>
<tr>
<tr>
-
<dr>2</dr>
+
<td>2</td>
-
<dr>Delta cheY 2</dr>
+
<td>Delta cheY 2 cm</td>
-
<dr>5 ug/ml</dr>
+
<td>5 ug/ml</td>
</tr>
</tr>
<tr>
<tr>
-
<dr>3</dr>
+
<td>3</td>
-
<dr>cheY negative control</dr>
+
<td>cheY negative control</td>
-
<dr>5 ug/ml cm</dr>
+
<td>5 ug/ml cm</td>
</tr>
</tr>
<tr>
<tr>
-
<dr>4</dr>
+
<td>4</td>
-
<dr>Delta des 1</dr>
+
<td>Delta des 1</td>
-
<dr>5 ug/ml cm 5 ug/ml km</dr>
+
<td>5 ug/ml cm 5 ug/ml km</td>
</tr>
</tr>
<tr>
<tr>
-
<dr>5</dr>
+
<td>5</td>
-
<dr>Delta des 2</dr>
+
<td>Delta des 2</td>
-
<dr>5 ug/ml cm 5 ug/ml km</dr>
+
<td>5 ug/ml cm 5 ug/ml km</td>
</tr>
</tr>
<tr>
<tr>
-
<dr>6</dr>
+
<td>6</td>
-
<dr>Delta des negative control</dr>
+
<td>Delta des negative control</td>
-
<dr>5 ug/ml cm 5 ug/ml km</dr>
+
<td>5 ug/ml cm 5 ug/ml km</td>
</tr>
</tr>
</table>
</table>

Latest revision as of 10:08, 2 August 2013

Mirjam

Plates for the transformation of ΔDes, ΔCheY and ΔDes/ΔCheY did not show any colonies. The plates for eYFP showed some colonies on the plates with the transformation to E.coli. The plates with RFP and the ligation of Pdes/Chey did not show any colonies. Also the plates with a transformation of GFP240 and GFP0840 showed colonies.
Inoculated the colonies into LB with ampicillin to perform a miniprep later on. Miniprep analysis is done and the products are used to perform a restriction digestion. This showed that one of the eYFP colonies is a correct transformant. This transformant is plated on LB agar plates with IPTG and Amp resistance.
Made a new restriction digestion for RFP, eYFP and BBa_k823823 + LacI with EcoRI and PstI.
Inoculated B.subtilis colonies for transformation.
Made a restriction for Pdes with EcoRI and BamHI, for CheY with BamHI and PstI and for BBa_k823823 with EcoRI and PstI.
Heat inactivated the restriction digestions of Des Up, Tet, Des down, CheY up, spec, CheY down and Pdes.
Did a PCR clean up for CheY and BBa_k823823.
Did a ligation reaction for Des Up, Tet and Des down as well as a ligation reaction for CheY up, spec and CheY down. Heat inactivated these reactions and did a transformation to B.subtilis.
Made a ligation reaction for Pdes and CheY and heat inactivated the reaction. This ligation reaction is ligated into BBa_k823823. Also this ligation is heat inactivated and the reaction is transformed to E.coli.
Ligation reactions RFP and eYFP with BBa_k823823 + LacI are made and heat inactivated. Then they are transformed into E. coli.
Because the B.subtilis culture didn't grow that well, it is decided to plate a fresh stock on agar. If todays reaction fails. There is another chance tomorrow.

Chaline

Made a miniprep of eYFP and RFP to get a higher concentration.
Did a gel purification to obtain purified fragments of eYFP, RFP and backbone BBa_k823823 + LacI.

Sebas

Sander

Made a plasmid purification of GFP BBa_E0240 and got a concentration of 21,5 and also did a purification of GFP BBa_E0840 and got a concentration of 13,5.(placed in temp box labeled as GFP 0240 and GFP 0840)

Claudio

The plates prepared yesterday show colonies. 4 colonies per plate are inoculated in LB + Ampicillin in order to isolate the plasmid on the day after and check for the presence of the desired insert.

To the tube, containing the purified BBa_K823023 + lacI digested with XbaI and PstI, 1µl SpeI is added and the tube is incubated for 1h at 37°C. After the treatment the backbone is ligated (3:1 insert:vector ratio) with BBa_E0840 and BBa_E0240 (already digested and purified yesterday with XbaI and PstI). The two ligation products are transformed into E. Coli DH5α and plated on LB + Ampicillin.

Bacillus Subtilis is inoculated in LB.

Inne & Sander

Did a inoculation of 2 colonies from the delta cheY plates and 2 colonies of the delta des Both plates were made yesterday.
Sample Strain Selection marker
1 Delta cheY 1 5 ug/ml cm
2 Delta cheY 2 cm 5 ug/ml
3 cheY negative control 5 ug/ml cm
4 Delta des 1 5 ug/ml cm 5 ug/ml km
5 Delta des 2 5 ug/ml cm 5 ug/ml km
6 Delta des negative control 5 ug/ml cm 5 ug/ml km