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From 2011.igem.org

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	==Saturday, September 3==		==Saturday, September 3==
	Afternoon lab work done by: Jim Mathew, Charlie Chung, Nancy Li		Afternoon lab work done by: Jim Mathew, Charlie Chung, Nancy Li
-		+	:'''Quantification of Purified DNA Samples via NanoDrop Spectrophotometry'''
-	Miniprep	+
-	-took concentrations of yesterday miniprep	+
	-all 260/280nm ratio were precisely near 1.8 indicting pure DNA		-all 260/280nm ratio were precisely near 1.8 indicting pure DNA
-		+	Concentration (ng/uL)
-	DNA                                                                                  Concentration (ng/uL)	+
	RFP 1 in PSB1C3 Backbone                    344.0		RFP 1 in PSB1C3 Backbone                    344.0

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Revision as of 05:11, 4 September 2011

August 28th - September 3rd

Sunday, August 28

Monday, August 29

Tuesday, August 30

Afternoon - Evening lab work done by: Claire Paduano, Youjin Cho, Maneesh Gupta, James Mathew, Charlie Chung

• Digestion Reactions

- specifics to be posted soon

• Agarose Gel Electrophoresis

- specifics to be posted soon

• Gel Extraction and Purification of DNA

- specifics to be posted soon (including some issues in cutting out the insert band vs. backbone band)

• NanoDrop Spectrophometry for Purified DNA Quantification

- specific [ ]s to be posted soon

• Ligation Reactions

- specifics to be posted soon

Wednesday, August 31

Afternoon/Evening Lab work done by: Claire Paduano and Maneesh Gupta

• Looked at ATTO590 coated chip from Aug 27th under fluorescent microscope: channels still fluorescent

Photo to be posted soon

Streptavidin coating and Fluorescent Probes

Procedure to be posted soon

Flow experiments

Details and photos to be posted soon

Thursday, September 1

Morning lab work done by: Claire Paduano and Nancy Li

• Coated three chips with streptavidin

Streptavidin coating

• Protocol from Gleghorn et al: http://www.ncbi.nlm.nih.gov/pubmed/20024046

1) 45 min in 4% (by volume) MPTMS in ethanol

2) 20 min in 1mM GMBS

3) 45 min in 25ng/mL NeutrAvidin in PBS

Evening lab work done by: Maneesh Gupta

• Did continuous flow experiments to test resilience of biotin-avitin binding
• Used same exposure (83.33) in all images taken

Friday, September 2

Afternoon lab work done by: Charlie Chung

• Miniprep DNA purification of cultures containing pSB1C3 vector backbone with (RFP+Avi-Tag), (vioA+Avi-Tag), (vioB+Avi-Tag), and (vioE+Avi-Tag) inserts -- in preparation for iGEM BioBrick submission

Saturday, September 3

Afternoon lab work done by: Jim Mathew, Charlie Chung, Nancy Li

Quantification of Purified DNA Samples via NanoDrop Spectrophotometry

-all 260/280nm ratio were precisely near 1.8 indicting pure DNA

                                                                                Concentration (ng/uL)

RFP 1 in PSB1C3 Backbone 344.0 RFP 2 in PSB1C3 Backbone 256.4 RFP 3 in PSB1C3 Backbone 230.4 VIOA 1-1 in PSB1C3 Backbone 205.9 VIOA 1-2 in PSB1C3 Backbone 494.3 VIOA 1-3 in PSB1C3 Backbone 370.8 VIOA 2-1 in PSB1C3 Backbone 892.5 VIOA 2-2 in PSB1C3 Backbone 203.8 VIOA 2-3 in PSB1C3 Backbone 206.0 VIOB 1-1 in PSB1C3 Backbone 503.9 VIOB 1-2 in PSB1C3 Backbone 186.1 VIOB 1-3 in PSB1C3 Backbone 398.8 VIOB 2-1 in PSB1C3 Backbone 868.9 VIOB 2-2 in PSB1C3 Backbone 145.6 VIOB 2-3 in PSB1C3 Backbone 174.6 VIOE 1 in PSB1C3 Backbone 266.2 VIOE 2 in PSB1C3 Backbone 552.9 VIOE 3 in PSB1C3 Backbone 400.7

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• DpnI Digestion of PCR Reaction Product

- PCR with specially designed primers to delete the iGEM restriction enzyme cut sites, which may have been forming too much distance between the ribosome binding site and the start codon of our gene

- first half of the forward primer consists of the base pairs flanking the deletion region to the left, second half of the forward primer consists of the base pairs flanking the deletion region to the right

- when the primer anneals to the template, it is complementary to the nucleotides on either side of the deletion region and pinches them in, forcing the deletion region to loop out without a complementary strand

• Transformation via Electroporation

- (RFP+Avi-Tag) into DH5α and MC400

- (vioA+Avi-Tag) into DH5α and MC400

- (vioB+Avi-Tag) into DH5α and MC400

- (vioE+Avi-Tag) into DH5α and MC400

• Plating of Transformed Bacteria

- Ampicillin-treated plates are incubating at 37°C in Weill Hall