Minnesota/18 June 2008

From 2008.igem.org

(Difference between revisions)

Revision as of 15:03, 27 June 2008

1. “Paper Punches”: Transfer DNA/plasmid of chosen gene from iGEM paper into 1.5 ml tube.

2. Assign numbers to genes/plasmids:

Number	Plasmid/gene	Resistance type
1	Tet R	Ampicillin Res.
2	YFP	Ampicillin Res.
3	MCherry	Ampicillin Res.
4	P22 MNTR	Kanamycin Res.
5	P22 MNTC	Kanamycin Res.
6	P22 CIIC	Ampicillin Res.
7	LAMBDA CIR	Ampicillin Res.
8	EYFP	Ampicillin Res.
9	P22 C2R	Ampicillin Res.
10	GFP	Ampicillin Res.
11	TERMINATOR	Amp. and Kan. Res.
12	RBS	Ampicillin Res.
13	LAMBDA CIC	Ampicillin Res.
14	LAC I	Ampicillin Res.

3. “Transformation”: Transformation means taking the gene/plasmid from the biobrick paper and transforming it to competent cells. Procedure: 2 microliters(uL) of plasmid DNA in TE was added to 50 uL thawed dh5alpha chemocompetent cells in a pre-chilled 1.5 mL tube. Cells were allowed to incubate for approximately 30 minutes on ice; then heat shocked @ 50 Celsius in a H20 bath for 60 seconds with agitation/mixing by flicking tubes @ every 20 second interval. Tubes were placed on ice for 2 minutes, then cells were transferred to 2mililiter glass culture tubes containing 500uL of 2xTy broth pre-warmed @ 37 Celsius. Cells were allowed to recover @ 37 Celsius with shaking @ 225 rpm (rotation-per-minute) for 2 hours. After this, cells were plated on LB plates containing antibiotic (ampicillin or kanamycin; determined by resistance gene in plasmid). Plates were incubated overnight for approximately 14 hours.

4. Steps of making antibiotic LB plate: spray hood area clean w/ 80% ethanol  turn on vent in hood so microbes are sucked up and away from plates  light a fire to kill airborne bacteria  using a pipet, measure ___ of antibiotic into liquid LB, mix, and eye ball pouring  use extra safety measures to prevent bacterial contamination since LB is nutrient rich.

5. General lab steps went through for the day: Cut out DNA paper from “Biobricks” section of iGEM notebook (sterilize scalpel by dipping into 10% bleach, water, then ethanol)  Place papers in individual 1.5 mL tube  Add 5uL of warmed TE for 20 minutes  Add 2uL of DNA in TE and 50uL of thawed dh5alpha competent cells  Allows DNA to enter competent cells (ice & heat)  transfer competent cells w/ DNA to 2xTy broth  500uL 2xTy broth used each placed @ 37C  Make agar/LB plate by mixing liquid LB w/ antibiotic (do this so no other cells can grow and contaminate the plate – only competent cells w/ biobrick DNA grows).

6. Biobrick numbers for genes/plasmids:

	Gene	Biobrick
	GFP	E0040
	RFP	J04051
	RBS	B0032
	Terminator	B1006
	Lambda CI	R0051, C0051
	P22 CII	R0053, C0053
	P22 mnt	R0072, C0072
	Tet R	R0040
	Lac I	R0010

- Biobrick website: parts.mit.edu ***

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 {|
-|June 18, 2008
 |-
 |1.	“'''Paper Punches'''”: Transfer DNA/plasmid of chosen gene from iGEM paper into 1.5 ml tube.
 |-
-|2.	'''Assign numbers to genes/plasmids''':
+|2.	'''Assign numbers to genes/plasmids:'''
 |}
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 |5.	'''General lab steps went through for the day''': Cut out DNA paper from “Biobricks” section of iGEM notebook (sterilize scalpel by dipping into 10% bleach, water, then ethanol)  Place papers in individual 1.5 mL tube  Add 5uL of warmed TE for 20 minutes  Add 2uL of DNA in TE and 50uL of thawed dh5alpha competent cells  Allows DNA to enter competent cells (ice & heat)  transfer competent cells w/ DNA to 2xTy broth  500uL 2xTy broth used each placed @ 37C  Make agar/LB plate by mixing liquid LB w/ antibiotic (do this so no other cells can grow and contaminate the plate – only competent cells w/ biobrick DNA grows).
 |-
-|6.	'''Biobrick numbers for genes/plasmids''':
+|6.	'''Biobrick numbers for genes/plasmids:'''
 |}