"

From 2010.igem.org

Revision as of 15:29, 12 August 2010 (view source) Mvoges (Talk | contribs) ← Older edit		Revision as of 15:47, 12 August 2010 (view source) Mvoges (Talk | contribs) (→Assembly of reference construct) Newer edit →
Line 82:		Line 82:
	====Assembly of reference construct====		====Assembly of reference construct====
		+
		+	=====Method 1=====
		+
		+	<i>Hypothesis II</i>
		+	The transformants that were replated on kanamycin plates produced colonies, indicating that hypothesis II was invalid.
		+
		+
		+	=====Method 4=====
		+
		+	The obtained colonies on ampicillin plates were screened for GFP fluorescence, those that fluoresced were inoculated in 5 mL of LB medium for over night growth.

---

Revision as of 15:47, 12 August 2010

Contents 1 Lab work 1.1 Ordered DNA 1.2 Alkane degradation 1.3 Salt Tolerance 1.4 Emulsifier 1.5 Characterisation of Anderson RBS sequences 1.5.1 Assembly of reference construct 1.5.1.1 Method 1 1.5.1.2 Method 4

Lab work

Ordered DNA

The transformations of 19 July containing the different ligations gave colonies (~5 per plate). We picked 5 colonies per plate and performed a colony PCR.

Alkane degradation

Unfortunately there were no transformants on yesterday's plates. This is most likely due to the fact that the ligation didn't work with the small pieces of DNA of the RBSs. Next plan is to cut open the RBS plasmid without removing the RBS (with SpeI and PstI) and insert the gene in this plasmid. We will try this tomorrow.

Salt Tolerance

The overnight ligation was transformed using the standard method and grown up overnight at 37 degrees C.

Emulsifier

There were small colonies on the plates. Pieter picked seven and performed colony PCR on them.

Lane Description

#	Description	Expected lenght (bp)	Primer	Status
M1	EZ Ladder	n/a	n/a	n/a
1	B0032 (control)	220	G00100 + G00101	✓
2	Transformant #1 of ligation mix R0011-B0032	300	G00100 + G00101	✗
3	Transformant #2 of ligation mix R0011-B0032	300	G00100 + G00101	✗
4	Transformant #3 of ligation mix R0011-B0032	300	G00100 + G00101	✗
5	Transformant #4 of ligation mix R0011-B0032	300	G00100 + G00101	✗
6	Transformant #5 of ligation mix R0011-B0032	300	G00100 + G00101	✗
7	Transformant #6 of ligation mix R0011-B0032	300	G00100 + G00101	✗
8	Transformant #7 of ligation mix R0011-B0032	300	G00100 + G00101	✗

Unfortunately all other lanes were empty or the same as the negative control. So from this gel we conclude that the ligation has failed.

Characterisation of Anderson RBS sequences

Assembly of reference construct

Method 1

Hypothesis II The transformants that were replated on kanamycin plates produced colonies, indicating that hypothesis II was invalid.

Method 4

The obtained colonies on ampicillin plates were screened for GFP fluorescence, those that fluoresced were inoculated in 5 mL of LB medium for over night growth.