"

From 2011.igem.org

Revision as of 16:19, 28 July 2011 (view source) Cc745 (Talk | contribs) (→Wednesday) ← Older edit		Revision as of 16:22, 28 July 2011 (view source) Cc745 (Talk | contribs) (→Wednesday) Newer edit →
Line 26:		Line 26:
	Lab work done by: James Mathew		Lab work done by: James Mathew
	*Set up ligation reaction of pZE-12 backbone with the primer dimer insert.		*Set up ligation reaction of pZE-12 backbone with the primer dimer insert.
-	::1. Digestion of Backbone	+	::1. <u>Digestion of Backbone</u>
	:::- 26 ul Backbone plasmid = 2 ug DNA		:::- 26 ul Backbone plasmid = 2 ug DNA
	:::- 5 ul Buffer #4 (optimal for SphI-HF and ClaI-HF)		:::- 5 ul Buffer #4 (optimal for SphI-HF and ClaI-HF)
Line 35:		Line 35:
	::::*left in water bath for 30 minutes		::::*left in water bath for 30 minutes
-	::2. Ran Digestion Product through gel for 30 minutes at 120 V.	+	::2. <u>Ran Digestion Product through gel for 30 minutes at 120 V.</u>
-	:::Lane 1: 2 ul 1kb Ladder + 2 ul 6X Dye + 8 ul H20	+	::::Lane 1: 2 ul 1kb Ladder + 2 ul 6X Dye + 8 ul H20
-	:::Lane 2: 25 ul digestion reaction + 5 ul 6X Dye	+	::::Lane 2: 25 ul digestion reaction + 5 ul 6X Dye
-	:::Lane 3: 25 ul digestion reaction + 5 ul 6X Dye	+	::::Lane 3: 25 ul digestion reaction + 5 ul 6X Dye
-	::3. Gel Purification using Qiagen Kit	+	::3. <u>Gel Purification using Qiagen Kit</u>
-	::4. NanoDrop of Samples	+	::4. <u>NanoDrop of Samples</u>
-	:::Concentration:	+	::::Concentration:
-	:::Label:	+	::::Label:
-	::5. Ligation Reaction	+	::5. <u>Ligation Reaction</u>
-	:::Label:	+	::::Label:
	*Ligation done overnight for transformation on 7/21/11		*Ligation done overnight for transformation on 7/21/11

---

Revision as of 16:22, 28 July 2011

July 17th - July 23rd

Sunday

Lab work done by: Alyssa Henning & Bill Jo

• Successfully miniprepped 1 sample of GFP + avitag (the second sample got messed up)
• Ran a gel on the RFP that was PCRed on Saturday. We also cut out the bands.
• Sean will innoculate 6 tubes of GFP-containing bacteria--1 tube for us, and 5 tubes for CURIE.

Monday

Lab work done by: Charlie Chung & Youjin Cho

• Gel purified 2 RFP PCR samples from Friday.
• Miniprepped 2 samples of GFP + avitag.

Tuesday

Lab Work Done By: James Mathew

• Submitted GFP+avitag genes for synthesis
• Submitted pZE-12 backbone for subcloning of light sensor

Wednesday

Lab work done by: James Mathew

• Set up ligation reaction of pZE-12 backbone with the primer dimer insert.

1. Digestion of Backbone

- 26 ul Backbone plasmid = 2 ug DNA

- 5 ul Buffer #4 (optimal for SphI-HF and ClaI-HF)

- 1 ul SphI & 1 ul ClaI

- 16.5 ul H20

• left in water bath for one hour

- 1 ul CIAP added to digestion reaction

• left in water bath for 30 minutes

2. Ran Digestion Product through gel for 30 minutes at 120 V.

Lane 1: 2 ul 1kb Ladder + 2 ul 6X Dye + 8 ul H20

Lane 2: 25 ul digestion reaction + 5 ul 6X Dye

Lane 3: 25 ul digestion reaction + 5 ul 6X Dye

3. Gel Purification using Qiagen Kit

4. NanoDrop of Samples

Concentration:

Label:

5. Ligation Reaction

Label:

• Ligation done overnight for transformation on 7/21/11

Thursday

Lab work done by: James Mathew

• miniprepped Vio operon (Cambridge 2009)

Lab work done by: Youjin Cho, Charlie Chung, and Alyssa Henning

• Reconstituted VioA, VioB, and VioE forward and reverse primers
• Set up PCR for VioA, VioB, and VioE
• Aborted transformation of pZE-12 plasmid + avitag primer dimer because reverse primers for avitag were incorrect.
• Redesigned reverse primers for avitag to order on Friday

Friday

Lab work done by: James Mathew & Claire Paduano

• PCR gel purification of VioA, VioB, and VioE

Saturday

Lab work done by: James Mathew & Claire Paduano

• Digestion of VioA, VioB, VioE, GFP, and RFP in preparation for ligation with corrected backbone and avitag primer dimer