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1. 10:00 -- Plates ,which are transformed, were taken from incubation and put into +4 C.
2. 11:30 –- Plasmid isolation of backbone ( 4 tane)
They were code as BB-10, BB11, BB12, BB13
3. 12:00 -- Electrophrose of LUXR-RBS, Term B0010, Term B0012 and RBS because nanodrop results of them were very high.
4. 16:30 -- Digestion of LUXR-RBS
LUXR-RBS EcorI and SpeI = Buffer R – 4 fold excess of speI ( commercial)
However, we used fast digest. Same buffer is used with EcorI and SpeI in fast digestion process. 1 microgram of DNA was used. 14 µl water- nuclease free 2 µl buffer 1.63 µl DNA 1 µl EcorI + 1 µl speI Wait for 1 hour at 37 C. Results: Only Backbone was cut.
5. Preparation of Agorose gel for ligation.
while loading to gel; 20 µl plasmid + 4 µl loading dye (no water) 5 µl Ladder