Team:Glendale CC AZ/igem.org

From 2013.igem.org

(Difference between revisions)
(Undo revision 49261 by Pepper360 (talk))
(Replaced content with "{{:Team:Glendale_CC_AZ/test}}")
 
Line 1: Line 1:
{{:Team:Glendale_CC_AZ/test}}
{{:Team:Glendale_CC_AZ/test}}
-
 
-
==Alkaline Lysis Plasmid Miniprep==
 
-
 
-
 
-
 
-
'''Purpose:''' To isolate plasmid DNA from transformed cells.
 
-
 
-
 
-
'''Reagents:'''
 
-
 
-
*Lysis solution (50mM glucose, 25mM TrisHCl pH 8, 10 mM EDTA)
 
-
 
-
: 9.0mL dH2O
 
-
 
-
: 0.25mL 1M TrisHCl pH 8
 
-
 
-
: 0.25mL 0.5 M Na2EDTA
 
-
 
-
: 0.50mL 20% glucose
 
-
 
-
*SDS/NaOH (prepare fresh)
 
-
 
-
: 880ul dH2O
 
-
 
-
: 100ul 10% SDS
 
-
 
-
: 20ul 10M NaOh
 
-
 
-
*Acetate solution
 
-
 
-
: 60mL 5M potassium acetate
 
-
 
-
: 11.5mL glacial acetic acid
 
-
 
-
: 28.5mL dH2O
 
-
 
-
 
-
'''Materials:'''
 
-
 
-
*1 colony of transformed cells
 
-
 
-
*5mL LB/chloramphenicol media
 
-
 
-
*Bucket with ice
 
-
 
-
*Microcentrifuge
 
-
 
-
*Microcentrifuge tubes
 
-
 
-
*Micropipette
 
-
 
-
*Disposable tips
 
-
 
-
*200ul lysis solution
 
-
 
-
*400ul SDS/NaOH (made fresh)
 
-
 
-
*300ul acetate solution
 
-
 
-
*1000ul isopropanol
 
-
 
-
*400ul 70% ethanol
 
-
 
-
*Kimwipes
 
-
 
-
*100ul TE
 
-
 
-
 
-
'''Procedure:'''
 
-
# Inoculate 5ml of LB/antibiotic (chloramphenicol) with a single colony of transformed cells. Incubate overnight at 37ºC with shaking.
 
-
# '''Optional''': Keep 2mL of the bacterial culture for indexing. Pipet 1.5mL of culture into a microcentrifuge tube, and microcentrifuge at 10,000 rpm for 1 minute. Discard the supernatant, and add the remaining 1.5mL of culture to the same microcentrifuge tube. Centrifuge the tube for 1 more minute at 10,000 rpm and discard supernatant one more time.
 
-
# Resuspend the pellet in 200ul of lysis solution. Incubate at room temperature for 5 minutes.
 
-
# Add 400ul of SDS/NaOH (made fresh) and invert 3-6 times. Incubate on ice for 5 minutes, solution will become clear and viscous.
 
-
# Add 300ul of acetate solution, gently mix for a few seconds, and incubate on ice for 10 minutes.
 
-
# Centrifuge for 3 minutes at 10,000 rpm in microcentrifuge.'''Optional''': Adjust microcentrifuge time depending on amount of cell growth. 
 
-
# Decant supernatant into a clean microcentrifuge tube; avoid the loose, viscous pellet.
 
-
# Add 1 volume of isopropanol and invert tube 3-6 times. Incubate at room temperature for 10 minutes to precipitate DNA.
 
-
# Centrifuge at 10,000 rpm for 10 minutes. Orient hinge to the same direction in case pellet is hard to see.
 
-
# Discard supernatant, removing residual liquid with pipet tip. Add 200-400ul 70% ethanol, agitate pellet gently and centrifuge at 10,000 rpm for 10 min to precipitate pellet.
 
-
# Discard supernatant, being careful to avoid losing the pellet. Let rest at room temperature with the lid open or inverted on a Kimwipe to evaporate residual alcohol.
 
-
# Dissolve pellet in just enough TE to dissolve the DNA, approx. 50-55ul. '''Optional''': Let pellet hydrate overnight.
 

Latest revision as of 23:39, 31 July 2013