Team:Paris/August 6
From 2008.igem.org
(→Digestion) |
(→Digestion) |
||
Line 57: | Line 57: | ||
* 0,25 µL BSA | * 0,25 µL BSA | ||
* 1 µL '''EcoRI''' | * 1 µL '''EcoRI''' | ||
- | <br> Incubation 1h30 at 37°C | + | <br> Incubation 1h30 at 37°C |
- | <br> Incubation 1h30 more after adding 1 µL of '''SpeI''' | + | <br> Incubation 1h30 more after adding 1 µL of '''SpeI''' |
<br> Electrophoresis of the digested J61002 plasmid | <br> Electrophoresis of the digested J61002 plasmid | ||
<br> Excision, dissolution and purification of the band of interest using the Wizard SV Gel and PCR Clean-Up System (Promega). | <br> Excision, dissolution and purification of the band of interest using the Wizard SV Gel and PCR Clean-Up System (Promega). | ||
<br> Purification of the digestion products of the PCR products using the same kit. | <br> Purification of the digestion products of the PCR products using the same kit. |
Revision as of 15:41, 6 August 2008
Assay to clone different flagella gene promotors into the J61002 plasmidWe have to digest (by EcoRI and SpeI) the PCR products of yesterday (amplification of pFlgA (MP124), pFlgB (MP125), pFlhB (MP126) and pFlhDC (MP127) in order to clone them into the J61002 plasmid (that we must extract and then digest by EcoRI and SpeI). Plasmid extractionThe J61002 plasmid was extracted from overnight bacteria culture using the QIAspin Miniprep Kit (QIAGEN).
Assay to quantify DNAIn order to quantify the DNA contained either in the Miniprep product of MP123 or in the PCR products of MP124, MP125, MP126 and MP127 previously purifed yesterday by the QIAquick Gel Extraction Kit, we carried out an electrophoresis assay in a 1,5% agarose gel.
Results : The MP123 plasmid is clearly visible but the PCR products (purified by QIAquick Gel Extraction) aren't. There might be a problem during the purification step using the QIAquick Gel Extraction. Whatever, we still go on with the digestion. Besides the MP123 sample (J61002 plasmid) was quantified using a Bio Photometer (Eppendorf).
DigestionFor each sample (MP123 and PCR products of MP124, MP125, MP126 and MP127):
|