Team:Heidelberg/Notebook/Sensing Group/Notebook/1stweek

(Difference between revisions)

Revision as of 10:24, 26 October 2008

Preparations were done at the end of the week before

Transformation of pDK48 and pTrc99a in E. coli DH5a (0.5 µl plasmid-DNA + 50 µl competent cells)
Glycerol-stock of Vibrio harveyi BB120, BB886, mm30, BB178, BB125
picked pTrc99a and pDK48 cultures and inoculated in 3 ml LB overnight
V. harveyi cultured on LB+-Agar plate
MiniPrep of pTrc99a and pDK48 from DHha

LuxP colony PCR

LuxQ, LuxS PCR

PCR of LuxP from V. harveyi colony
Gel Purification of LuxP eluted in 30 µl H₂O
Digestion of LuxP with SalI and NotI of pDK48 and LuxP-Gel-Purification product: 2 µl NEB-Buffer 3 + 0.5 µl SalI/NotI + 10 µl DNA + 7 µl H₂O
Ligation with vector:insert ratio 1:3 and 1:1

5 colonies of LuxQ,S,P transformation (1:1 ligation) picket and used for colony-PCR
Colony-PCR to check for insert

Colony PCR of LuxQ, LuxS and LuxP

PCR to confirm insert from Minipreps. LuxQ no. 3 seems to be posivite. For LuxS and LuxP all samples are positive.

LuxQ colony PCR of 10 new colonies.

@@ Line 158: / Line 158: @@
 * Miniprep of samples Q#1-5, S#1-5, P#1-5
 * PCR from Minipreps to confirm inserts
-[[Image:HD 080807-LuxQ P S miniPCR.png|thumb|500px|PCR to confirm insert from Minipreps]]
+[[Image:HD 080807-LuxQ P S miniPCR.png|center|thumb|500px|PCR to confirm insert from Minipreps. LuxQ no. 3 seems to be posivite. For LuxS and LuxP all samples are positive.]]
+* 10 new colonies of LuxQ-plate picked and checked via colony-PCR
+[[Image:HD_080807-LuxQ_ColonyPCR.png|center|thumb|500px|LuxQ colony PCR of 10 new colonies.]]
 == Friday, 08/08/2008 ==