Generating and Amplifying Adenoviral Constructs - Revision history

Irith at 20:56, 11 July 2012

2012-07-11T20:56:45Z

← Older revision		Revision as of 20:56, 11 July 2012
Line 80:		Line 80:
	# Add virus sample (up to 2.5 ml)		# Add virus sample (up to 2.5 ml)
	# Elute with 5 ml PBS, discard first 20 drops and collect 10 drop fractions into 10 tubes.		# Elute with 5 ml PBS, discard first 20 drops and collect 10 drop fractions into 10 tubes.
	# OD 260 fractions in nanodrop, virus is in middle fractions (5-7 in my extraction). Good virus yield is >3 OD.		# OD 260 fractions in nanodrop, virus is in middle fractions (5-7 in my extraction). Good virus yield is >3 OD.
			# dilute to 1 OD/ml
	# Add 10% glycerol and store at -80.		# Add 10% glycerol and store at -80.

Irith: /* Purification of Adenovirus */

2011-03-23T19:18:27Z

Purification of Adenovirus

← Older revision		Revision as of 19:18, 23 March 2011
Line 74:		Line 74:
	#Add an equal volume of 2X Virus storage buffer and store at -80		#Add an equal volume of 2X Virus storage buffer and store at -80
	#Titer the virus using a kit.		#Titer the virus using a kit.


			===desalting of CsCl purified adenovirus using PD-10 desalting columns (17-0851-01 GE Healthcare)===
			# Cut column, discard storage solution and wash X5 with ~5ml PBS
			# Add virus sample (up to 2.5 ml)
			# Elute with 5 ml PBS, discard first 20 drops and collect 10 drop fractions into 10 tubes.
			# OD 260 fractions in nanodrop, virus is in middle fractions (5-7 in my extraction). Good virus yield is >3 OD.
			# Add 10% glycerol and store at -80.

			===Tail Vein Injection===
			# Insert mouse to restriction device. Carefully warm tail with lamp/warm water, inject 0.1 OD/mouse (0.15 for HFD mouse) using insulin syringe.
			# Injected mice are housed in biohazard mouse room for 3 days (need bright-pink barcode stickers and water bottles).
			# Adenovirus is detected in liver for at least 2-3 weeks, shRNA has maximal effect at 4-6 days, usually gone in 10 days.

Davebrid: changed beckman tube order number

2010-09-14T20:02:58Z

changed beckman tube order number

@@ Line 12: / Line 12: @@
 *100% ethanol
 *2 mm electroporation cuvette
 ==Protocol==
@@ Line 27: / Line 30: @@
 #Add 8 uL resuspended linearized plasmid to 20 uL electrocompetent cells
 #Carefully transfer to ice cold 2 mm electroporation cuvette avoiding bubbles and keeping on ice
-#Electroporate at '''pulse at 2,500 V, 200 O and 25 mF'''
+#Electroporate at '''2,500 V, 200 O and 25 mF'''
 #Resuspend in 500 uL LB and plate on 2-5 LB/Km plates
 #Pick 10-20 of the smallest colonies the next day and grow in LB/Km broth
 #Miniprep clones.  Digest 10 uL with PacI and check size by running out both digested and undigested clones on a 0.8% agarose gel.  Correct recombinants usually yield a large fragment (approximately 30 kb) and a smaller fragment of 3.0 or 4.5 kb.
 #Retransform and amplify correct clone(s).

Davebrid: Added linearlization and transformation protocols

2010-09-14T18:53:49Z

Added linearlization and transformation protocols

New page

__NOTOC__
[[Category:shRNA]][[Category:Adenovirus]][[Category:Transduction]][[Category:Knockdown]][[Category:Molecular Biology]]

==Materials==
*Targetting Construct in pAdtrack vector or the like (see [[ Preparing an Adenoviral shRNA Clone ]])
*PmeI restriction enzyme (or other enzyme as required for linearization.
*PacI restriction enzyme
*Electrocompetent BJ5183-AD-1 cells (Agilent Cat#200157; or prepare yourself)
*7.5 M ammonium acetate
*20mg/mL glycogen
*25:24:1 phenol/chloroform/isoamyl alcohol, pH 8.0
*100% ethanol
*2 mm electroporation cuvette

==Protocol==
see [http://www.coloncancer.org/adeasy/He,%20AdEasy%20-%20Nature%20Protocols,%202007.pdf Nature Protocols], [http://onlinelibrary.wiley.com/doi/10.1002/0471142905.hg1204s40/pdf Current Protocols in Human Genetics] protocols.
===Linearization and Purification of Shuttle DNA===
#Digest 0.2-0.5 ug plasmid DNA in 100 uL volume with 30-100U of PmeI overnight at 37C. If desired check a small amount by electrophoresis to ensure complete ligation.
#Add 100 uL ddH2O, 100 uL 7.5 M ammonium acetate and 2 uL 20 mg/ml glycogen.
#Add 300 uL 25:24:1 phenol/chloroform/isoamyl alcohol, pH 8.0.
#Remove top layer to clean tube and add 600 uL of 100% ethanol
#Centrifuge 5 min at 13 000 RPM
#Wash three times with 70% ethanol to remove residual salt.
#Dry and resuspend in 8 uL

===Transformation into BJ5183-AD-1 cells===
#Add 8 uL resuspended linearized plasmid to 20 uL electrocompetent cells
#Carefully transfer to ice cold 2 mm electroporation cuvette avoiding bubbles and keeping on ice
#Electroporate at '''pulse at 2,500 V, 200 O and 25 mF'''
#Resuspend in 500 uL LB and plate on 2-5 LB/Km plates
#Pick 10-20 of the smallest colonies the next day and grow in LB/Km broth
#Miniprep clones. Digest 10 uL with PacI and check size by running out both digested and undigested clones on a 0.8% agarose gel. Correct recombinants usually yield a large fragment (approximately 30 kb) and a smaller fragment of 3.0 or 4.5 kb.
#Retransform and amplify correct clone(s).