Team:Freiburg Bioware/NoteBook/Labjournal/October2

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146. labday 11.10.2010

cloning of lITR_CMV_betaglobin and lITR_phTERT_betaglobin into pSB1C3_CD

Investigator: Stefan

Comment: In order to test our GOI with another promotor, lITR_phTERT_beta-globin_mGMK_TK30 needs to be cloned upstream of hgH_rITR.


Vector name:
pSB1C3_CD clone 1 (P???)
pSB1C3_CD clone 2 (P???)

Insert name:
pSB1C3_lITR_CMV_beta-globin (P729)
pSB1C3_lITR_phTERT_beta-globin (P730)

Digestion:


components' volume CD clone 1 + 2 /µlvolume P729 /µl'volume P730 /µl
DNA 6 146
BSA (10x) 2 22
Buffer 4 (10x)2 22
Enzyme EcoI11 1
Enzyme XbaI1- -
Enzyme SpeI-11
H2O8- 8
Total volume (e.g. 15,20,25,30 µl) 20 20 20


Gel:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt



File:New.jpg



Gel extraction:
Was performed according to protocol.


T4 Ligation:

ligation name 728 + 670
volume of vector 2,35
volume of insert5,65
T4 ligase buffer (10x)1
T4 ligase 1


Transformation:
Was performed according to standard protocol using BL21 cells.

147. labday 12.10.2010: Example Example Example Example Example

148. labday 13.10.2010

149. labday 14.10.2010

150. labday 15.10.2010

151. labday 16.10.2010

152. labday 17.10.2010

153. labday 18.10.2010

154. labday 19.10.2010

155. labday 20.10.2010

=> Go to Labjournal October part 3 (labday 156 - 166 )