Team:Freiburg Bioware/NoteBook/Labjournal/October2

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(Colony PCR of p40_VP123_capins)
(Colony PCR of p40_VP123_capins)
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The PCR products were loaded on a 1% agarose gel. The results can be seen above in the gel picture: <br />
The PCR products were loaded on a 1% agarose gel. The results can be seen above in the gel picture: <br />
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<b>Result: Ee can see two things: The cloning of p40 to VP123 worked quiet well AND the Robust PCR Kit which was used for the first time worked as well.</b>
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<b>Result: We can see two things: The cloning of p40 to VP123 worked quiet well AND the Robust PCR Kit which was used for the first time worked as well.</b>
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[[Image: Freiburg10_ColonyPCR_p40VP123.png|thumb|center|400px]]
[[Image: Freiburg10_ColonyPCR_p40VP123.png|thumb|center|400px]]

Revision as of 20:08, 11 October 2010



Contents

146. labday 11.10.2010

Hannas colony PCR:

Investigator: Achim

Freiburg10 1110 testdigestionhanna1label.png
Freiburg10 1110 testdigestionhanna2label.png


All samples match the positive control!

Colony PCR of p40_VP123_capins

Investigator: Bea

Comment: Since the first attempt did not work,and no cells grew on the plate and the same ligation was transformed again into BL-21 and a lot of clones grew on the plate, I decided to perform a colony PCR in order to check several colonies and to inoculate at the same day for a Midi-Prep.

Protocol:

  • Primer used: O162
  • Primer used: O38
Freiburg10 ColonyPCR p40vp123 1110.PNG


The PCR products were loaded on a 1% agarose gel. The results can be seen above in the gel picture:

Result: We can see two things: The cloning of p40 to VP123 worked quiet well AND the Robust PCR Kit which was used for the first time worked as well.

Freiburg10 ColonyPCR p40VP123.png


cloning of lITR_CMV_betaglobin and lITR_phTERT_betaglobin into pSB1C3_CD

Investigator: Stefan

Comment: To produce another GOI for testing in cell culture, the cytosine deaminase needs to be assembled with lITR_promotor_betaglobin. In the next step hgH_rITR needs to be added.


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 /µl volume 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



Freiburg10 cloning 111010.jpg


CD clone 1 yielded to bands around 2500 bp to 3000 bp. Since the vector was cut only using EcoRI and SpeI, it was expected to be linearized, not to be cut into two fragments this size. Therefore, this sample was discarded and cloning was continued using CD clone 2.



Gel extraction:
Was performed according to protocol.


T4 Ligation:

ligation name 729 + CD cl2730 + CD cl2
volume of vector 3,672,82
volume of insert4,335,18
T4 ligase buffer (10x)11
T4 ligase 11


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 )