Team:Newcastle/24 August 2010

From 2010.igem.org

(Difference between revisions)
(Materials and Protocol)
Line 58: Line 58:
We are doing two digests for pGFPrrnB and ''yneA'':
We are doing two digests for pGFPrrnB and ''yneA'':
-
* Single digest with HinDIII;
+
* Single digest with HindIII;
-
* Double digest with EcoR1 and Nhe1.
+
* Double digest with EcoRI and NheI.

Revision as of 00:46, 28 October 2010

iGEM Homepage Newcastle University BacillaFilla Homepage Image Map

Contents

Miniprep for pGFPrrnB with filamentous cell part

Aims

The aim of this experiment is to obtain stock of the filamentous cell part in pGFP-rrnB for transformation into Bacillus subtilis 168.

Materials and Protocol

Please refer to qiagen minipreps and nanodrop spectrophotometer protocols.

Results

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 Tube 7 Tube 8 Tube 9 Tube 10 Tube 11 Tube 12
286.1 µl/ml 304.0 µl/ml 316.3 µl/ml 421.6 µl/ml 518.7 µl/ml 460.1 µl/ml 370.1 µl/ml 377.3 µl/ml 346.0 µl/ml 347.4 µl/ml 202.8 µl/ml 307.4 µl/ml

Table 1: Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.

Discussion

The concentration ranged from 286.1 µl/ml to 518.7 µl/ml. Therefore we have obtained high concentration of yneA in pGFPrrnB.

Single and Double Digestion of pGFPrrnB with yneA

Aims

The aim of this experiment is to check if the insert yneA has been inserted into vector pGFPrrnB.

Materials and Protocol

We are doing two digests for pGFPrrnB and yneA:

  • Single digest with HindIII;
  • Double digest with EcoRI and NheI.


Please refer to restriction digests and gel electrophoresis.

Discussion

Gel electrophoresis will be done on the 25th August, 2010.

Single digestion of pSB1C3

Aims

To linearise the vector pSB1C3. The linear vector will be used as a PCR template for Gibson cloning of the Subtilin immunity and rocF BioBricks.

Materials and Protocol

We are doing a single digest with HinDIII.

Please refer to restriction digests and gel electrophoresis.

Results

Gel electrophoresis results for digestion:

24.08.10.png

Figure 1: Gel electrophoresis of the amplified PCR products and restriction digest

  • Lane 1: 1 Kb ladder
  • Lane 2: digested (linearised) pSB1C3

Discussion and Conclusion

The digestion was successful — the correct band was seen. We can proceed to gel extraction.

Newcastle University logo.png    Newcastle cbcb logo.pngNewcastle Biomedicine logo.gif    Team Newcastle CEG logo.gif
Newcastle iww logo.jpg  UNIPV Pavia Logo.gif  Newcastle BBSRC.gif    Newcastle Genevision logo.png Newcastle WelcomeTrust.jpg
FaceBook Icon