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

(Difference between revisions)

Revision as of 20:28, 26 October 2008

Preparations were started 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 plate
MiniPrep of pTrc99a and pDK48 from DH5alpha

LuxP colony PCR

LuxQ, LuxS PCR

PCR of LuxP from V. harveyi colony with the primers LuxPc and LuxPd
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. 5min @ 94°C || 30s @ 94°C | 30s @ 58 °C | 2min @ 72°C || 10min @ 72°C | 4°C hold (30 cycles)

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.

Sequencing Results
LuxS: correct sequence
LuxP: all clones contain mutations, cloning has to be repeated
LuxQ: no insert in the picked colonies

@@ Line 137: / Line 137: @@
 [[Image:HD_080805-LuxP_PCR.png|thumb|150px|LuxP colony PCR]]
 <div style="float: right; clear:none">[[Image:HD 080805-LuxQ S PCR.png|right|thumb|200px|LuxQ, LuxS PCR]]</div>
-* PCR of LuxP from ''V. harveyi'' colony
+* PCR of LuxP from ''V. harveyi'' colony with the primers LuxPc and LuxPd
 * Gel Purification of LuxP eluted in 30 µl H<sub>2</sub>O
 * 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 H<sub>2</sub>O