Team:Wageningen UR/Journal/week12

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Biobrick BBa_J04450 was cloned and miniprepped.
Biobrick BBa_J04450 was cloned and miniprepped.
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''' Bricking Hepatitis B '''
+
''' Hepatitis B '''
-
Tuesday:
+
'''Tuesday:'''
-
* Digestion:
+
Digestion of
-
of the Hepatitis B PCR product (with pre- and suffix) with Spe1 and Pst1  
+
* Hepatitis B PCR product (with pre- and suffix) with Spe1 and Pst1
-
of BBa_J04500 (an IPTG inducible promoter with RBS) with Xba1 and Pst1  
+
* BBa_J04500 (an IPTG inducible promoter with RBS) with Xba1 and Pst1  
-
of BBa_PSB1K3.ml (linearized plasmid backbone) with EcoR1 and Pst1  
+
* BBa_PSB1K3.ml (linearized plasmid backbone) with EcoR1 and Pst1  
-
* PCR purification on the digest as a replacement for the heat inactivation
+
-
Wednesday:
+
PCR purification on the digest as a replacement for the heat inactivation
-
* Ligation:
+
-
of the Hepatitis B PCR product with BBa_J04500
+
-
of the Hepatitis B product with BBa_PSB1K3.ml
+
-
The ligations where done in duplo – once following the standard iGEM protocol and once using equimolar amounts of Hep B and the vector (DNA concentrations where measured by using NanoDrop)
+
-
* Electrotransformation in E.coli
+
-
* Grow transformed E.coli on plates containing kanamycin
+
-
A plasmid with a GFP coding device was used as a positive control (growing on a plate containing ampicillin)
+
-
Thursday:  
+
'''Wednesday:'''
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* Colony PCR
+
-
of 20 colonies transformed with the Hep B + BBa_J04500 construct
+
-
of 10 colonies transformed with the Hep B + BBa_PSB1K3.ml construct
+
 +
Ligation of
 +
* Hepatitis B PCR product with BBa_J04500
 +
* the Hepatitis B PCR product with BBa_PSB1K3.ml
 +
''The ligations where done in duplo – once following the standard iGEM protocol and once using equimolar amounts of Hep B and the vector (DNA concentrations where measured by using NanoDrop)''
-
Friday:
 
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* gel electrophoresis (1% agarose gel)  of the colony PCR samples
 
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colony PCR of 20 colonies transformed with the Hep B + BBa_J04500:
 
-
[[File:HepB colony PCR 19-7.jpg|200px]]
+
Electrotransformation of the 2 different constructs with DH5α
 +
* Grow transformed E.coli on plates containing kanamycin
 +
* A plasmid with a GFP coding device was used as a positive control (growing on a plate containing ampicillin)
 +
 +
 
 +
'''Thursday:'''
-
the colonies transformed with the Hep B + BBa_PSB1K3.ml construct did not have any Hep B inserts
+
Colony PCR
 +
* of 20 colonies transformed with the Hep B + BBa_J04500 construct
 +
* of 10 colonies transformed with the Hep B + BBa_PSB1K3.ml construct
 +
''out of 20 samples taken from the transformation with the HepB + BBa_J04500 construct – 7 turned out to have the right insert. The colonies transformed with the Hep B + BBa_PSB1K3.ml construct did not have any Hep B inserts''

Revision as of 15:52, 29 August 2012

week 12: 16 july - 22 july

Office work

Lab work

Biobrick BBa_J04450 was cloned and miniprepped.

Hepatitis B


Tuesday:

Digestion of

  • Hepatitis B PCR product (with pre- and suffix) with Spe1 and Pst1
  • BBa_J04500 (an IPTG inducible promoter with RBS) with Xba1 and Pst1
  • BBa_PSB1K3.ml (linearized plasmid backbone) with EcoR1 and Pst1


PCR purification on the digest as a replacement for the heat inactivation


Wednesday:

Ligation of

  • Hepatitis B PCR product with BBa_J04500
  • the Hepatitis B PCR product with BBa_PSB1K3.ml

The ligations where done in duplo – once following the standard iGEM protocol and once using equimolar amounts of Hep B and the vector (DNA concentrations where measured by using NanoDrop)


Electrotransformation of the 2 different constructs with DH5α

  • Grow transformed E.coli on plates containing kanamycin
  • A plasmid with a GFP coding device was used as a positive control (growing on a plate containing ampicillin)


Thursday:

Colony PCR

  • of 20 colonies transformed with the Hep B + BBa_J04500 construct
  • of 10 colonies transformed with the Hep B + BBa_PSB1K3.ml construct

out of 20 samples taken from the transformation with the HepB + BBa_J04500 construct – 7 turned out to have the right insert. The colonies transformed with the Hep B + BBa_PSB1K3.ml construct did not have any Hep B inserts


TuYV

  • Mach1 electrocompetent E. coli cells were transformed with last week's ligation mixtures. Results were unsatisfying.
  • 'Coat Protein + readthrough' gene amplicons were digested, ligated into pSB1K3 backbone and transformed into our electrocompetent Mach1 E. coli cells. After two attempts and a colony PCR, results were still unsatisfying.


PLRV

After a two week quest for PLRV infected plant material, we obtained infected potato leafs from the Dutch General Inspection Service for Agricultural seed and seed potatoes.


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