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Revision as of 09:36, 12 September 2012 (view source) Marnix (Talk | contribs) (→Lab work) ← Older edit		Revision as of 19:12, 13 September 2012 (view source) Lisamsonne (Talk | contribs) (→week 12: 16 july - 22 july) Newer edit →
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	Biobrick BBa_J04450 was cloned and miniprepped.		Biobrick BBa_J04450 was cloned and miniprepped.
-	''' Hepatitis B '''	+	''' HepB '''
		+	* digestion of the HepB core protein PCR product with pre- and suffix (from 11.July) and ligation into BBa_J04500 (an IPTG inducible promoter with RBS)as well as BBa_PSB1K3.ml (linearized plasmid backbone)
		+	* electro transformation of these constructs with DH5α
		+	* colony PCR (20 samples of the transformants containing HepB core protein + BBa_J04500; 10 samples of the transformants containing HepB core protein + BBa_PSB1K3.ml
		+	-> the ligation and transformation of the HepB core protein into the linear backbone (BBa_PSB1K3.ml) was not successful
		+	-> the colony PCR shows that we have 6 out of 20 colonies with the expected insert size (around 600bp)
-	'''Tuesday:'''
-	Digestion of	+	[[File:ColonyPCR_20July.png|200px]]
-	* 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	+
		+	''colony PCR 20.July''
-	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''

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Revision as of 19:12, 13 September 2012

week 12: 16 july - 22 july

Office work

Lab work

Biobrick BBa_J04450 was cloned and miniprepped.

HepB

• digestion of the HepB core protein PCR product with pre- and suffix (from 11.July) and ligation into BBa_J04500 (an IPTG inducible promoter with RBS)as well as BBa_PSB1K3.ml (linearized plasmid backbone)
• electro transformation of these constructs with DH5α
• colony PCR (20 samples of the transformants containing HepB core protein + BBa_J04500; 10 samples of the transformants containing HepB core protein + BBa_PSB1K3.ml

-> the ligation and transformation of the HepB core protein into the linear backbone (BBa_PSB1K3.ml) was not successful -> the colony PCR shows that we have 6 out of 20 colonies with the expected insert size (around 600bp)

colony PCR 20.July

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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