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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 ΔDes, ΔCheY and ΔDes/ΔCheY 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 ΔDes, ΔCheY and ΔDes/ΔCheY 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 | + | <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> | ||
| Line 42: | Line 51: | ||
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, 1µl SpeI is added and the tube is incubated for 1h at 37°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> DH5α and plated on LB + Ampicillin. | + | <br>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 <i>E. Coli</i> DH5α 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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<tr> | <tr> | ||
<td>2</td> | <td>2</td> | ||
| - | <td>Delta cheY 2</td> | + | <td>Delta cheY 2 cm</td> |
<td>5 ug/ml</td> | <td>5 ug/ml</td> | ||
</tr> | </tr> | ||
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 |
iGEM 2013 Groningen
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