Team:SDU-Denmark/labnotes6
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Methods: | Methods: | ||
protocos: | protocos: | ||
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Notes:<br> | Notes:<br> | ||
100 mL ON culture was made, cells were grown at 37 C in LB media with Chloramphenicol. | 100 mL ON culture was made, cells were grown at 37 C in LB media with Chloramphenicol. | ||
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Methods: Miniprep, Restriction digest, gel<br> | Methods: Miniprep, Restriction digest, gel<br> | ||
Protocols: MP1.2 [https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2], RD1.1 [https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1]. | Protocols: MP1.2 [https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2], RD1.1 [https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1]. | ||
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Notes:<br> | Notes:<br> | ||
50 mL of elution buffer was used. DNA concentrations after pooling were measured on NanoDrop to 206,2 ng/microL. | 50 mL of elution buffer was used. DNA concentrations after pooling were measured on NanoDrop to 206,2 ng/microL. | ||
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Methods: Restriction digest, gel<br> | Methods: Restriction digest, gel<br> | ||
Protocols: MP1.2 [https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2], gel. | Protocols: MP1.2 [https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2], gel. | ||
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Notes:<br> | Notes:<br> | ||
Due to the lack of old sample, restriction digest was performed using only 3,5 microL of miniprep produckt. 1,5 microL of H2O was added insted. EcoRI was used<br> | Due to the lack of old sample, restriction digest was performed using only 3,5 microL of miniprep produckt. 1,5 microL of H2O was added insted. EcoRI was used<br> | ||
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Methods: ON<br> | Methods: ON<br> | ||
protocos:CP.1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1] | protocos:CP.1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1] | ||
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Notes:<br> | Notes:<br> | ||
The new primers only contain the innermost restriction sites.<br> | The new primers only contain the innermost restriction sites.<br> | ||
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Methods: PCR<br> | Methods: PCR<br> | ||
protocos:CP.1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1] | protocos:CP.1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1] | ||
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Notes:<br> | Notes:<br> | ||
NinB2fw and NinaB2rv was used.<br> | NinB2fw and NinaB2rv was used.<br> | ||
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protocos:GFX purification from PCR - kit | protocos:GFX purification from PCR - kit | ||
<br><br> | <br><br> | ||
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One of the pooled tubes was eluted in 200µL, the others was eluted in 20µL<br> | One of the pooled tubes was eluted in 200µL, the others was eluted in 20µL<br> | ||
200µL nanodrop: 16,4 ng/µL<br> | 200µL nanodrop: 16,4 ng/µL<br> | ||
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Methods: Restriction Digest<br> | Methods: Restriction Digest<br> | ||
protocos:RD1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1] | protocos:RD1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1] | ||
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Notes:<br> | Notes:<br> | ||
+ | Restriction mixture:<br> | ||
+ | <html> | ||
+ | <head> | ||
+ | <meta content="text/html; charset=ISO-8859-1" | ||
+ | http-equiv="content-type"> | ||
+ | <title></title> | ||
+ | </head> | ||
+ | <body> | ||
+ | <table style="text-align: left; width: 100%;" border="1" | ||
+ | cellpadding="2" cellspacing="2"> | ||
+ | <tr> | ||
+ | <td>38 µL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>8 µL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4 µL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4 µL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>30 µL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | </body> | ||
+ | </html> | ||
+ | Gel was run with uncut controles:<br> | ||
+ | |||
+ | ==== Gel purification ==== | ||
+ | Date: 24/8<br> | ||
+ | Done by: Marie & Tommy<br> | ||
+ | Methods: gel purifikation<br> | ||
+ | protocos:GFX gel purifikation kit | ||
+ | Notes:<br> | ||
+ | Purifide products was Nanodroped:<br> | ||
+ | NinaB 1: 4,5 ng/µL<br> | ||
+ | NinaB 2: 1,15 ng/µL<br> | ||
+ | NinaB 3: 7,74 ng/µL<br> | ||
+ | PSB1C3: 25,66 ng/µL<br> | ||
+ | Nina B pooled: 4,5 ng/µL<br> | ||
+ | |||
+ | ==== Ligation ==== | ||
+ | Date: 24/8<br> | ||
+ | Done by: Marie & Tommy<br> | ||
+ | Methods: Ligation<br> | ||
+ | protocos:L1.3[https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.3] | ||
+ | Notes:<br> | ||
+ | 3 ligatons mixtures was made:<br> | ||
+ | 1:1 volumens 1 plasmid:5 insert | ||
+ | 1:3 volumens 1 plasmid:15 insert | ||
+ | 1:6 volumens 1 plasmid:30 insert |
Revision as of 11:34, 1 September 2010