Matt Hartings at 16:19, 4 January 2013

2013-01-04T16:19:25Z

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	==Objective==		==Objective==
	Prepping some work for the weeks ahead		Prepping some work for the weeks ahead

Matt Hartings at 16:18, 4 January 2013

2013-01-04T16:18:31Z

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	==Objective==		==Objective==
	Prepping some work for the weeks ahead		Prepping some work for the weeks ahead

Matt Hartings at 15:57, 4 January 2013

2013-01-04T15:57:13Z

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	==Objective==		==Objective==
-	~~Learn how to maintain an OpenWetWare Notebook.~~	+	Prepping some work for the weeks ahead

		+	==Cloning==
		+	===PCR===
		+	# I'm going to try to do the Mb cloning into pQE-80 myself. (May want to try [http://www.qiagen.com/products/cloning/pcrcloningsystem/qiagenpcrcloningkit.aspx pDrive Coning Vector] eventually)
		+	## The following protocol is taken from [http://www.its.caltech.edu/~bjorker/Protocols/cloning%20protocols%20by%20Astrid.pdf here]
		+	## PCR mix
		+	### 10X reaction Buffer
		+	#### 2.5μL
		+	### NTP's stock solution is 10mM (make 50μL of 2mM by diluting 10μL of stock)
		+	#### 2.5μL
		+	### 3' primer (25μM) (need to check my concentrations for rough estimates)
		+	#### 1.0μL
		+	### 5' primer (25μM) (need to check my concentrations for rough estimates)
		+	#### 1.0μL
		+	### DNA template (0.1μg/μL) (need to check my concentration for rough estimate)
		+	#### 1.0μL
		+	### Autoclaved water
		+	#### 16.5μL
		+	### Pfu polymerase
		+	#### 0.5μL
		+	## PCR cycles
		+	### 1' 94°C
		+	### 1' 55°C
		+	### 1' 72°C
		+	### Repeat previous three steps 3 times
		+	### 1' 94°C
		+	### 1' 60°C
		+	### 1' 72°C
		+	### Repeat previous three steps 12 times
		+	### 7' 72°C
		+
		+	===Purifying PCR product===
		+	# Run a 1% Gel
		+	## (25 mL TBE, 0.25g agarose)
		+	# Incubate gel in ethidium bromide solution for 30 minutes
		+	# Incubate gel in rinse solution for 30 minutes
		+	# Look for band on illuminator
		+	## If band exists, cut out of gel with razor
		+	# Follow protocol from Promega Wizard miniprep kit
		+	## melt the gel for 5-10' at 70°C
		+	## add 1mL purification resin and mix
		+	## put the resin in the column
		+	## wash with 2mL 80% isopropanol
		+	## spin 2' at 14000 rpm
		+	## air dry for 5'
		+	## add 50uL autoclaved water and incubate 2'
		+	## spin 30 seconds at 14000 rpm
		+
		+	===Restriction of PCR product===
		+	If you had a bright DNA band after PCR, use 20μL of the product. If you had a faint band, lyophilize the 50μL from the previous step and add 20μL water. The restriction enzymes that I need for [http://www.qiagen.com/literature/pqesequences/pqe8x.pdf pQE-80] are: BamHI and HindIII
		+
		+	# Start protocol first thing in the morning
		+	# Do the restriction in a total 50μL mix
		+	## normally choose the buffer that is good for both enzymes, but NEB says to do them [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/double_digests.asp#.UObxUo5QZuo sequentially]
		+	# Add 5μL HindIII buffer
		+	# Add 24μL water
		+	# Add 1μL HindIII
		+	# Incubate for day at 37
		+	# Add 1μL of 5M NaCl to buffer see [http://www.protocol-online.org/biology-forums/posts/5618.html discussion thread]
		+	# Add 1μL BamHI
		+	# Incubate overnight
		+	# Heat inactivate 20' at 65°C
		+	# (Don't know if I need to gel purify here)
		+
		+	===Restriction of vector===
		+	Run the cuts at the end of the day
		+	# cut 10μg vector in a 50μL mix
		+	## Add appropriate volume of DNA (check concentration)
		+	## Add 5μL HindIII buffer
		+	## Add appropriate volume of water to 49μL
		+	## Add 1μL HindIII
		+	## Incubate at 37°C overnight
		+	# Run a 1% Gel
		+	## (25 mL TBE, 0.25g agarose)
		+	# Incubate gel in ethidium bromide solution for 30 minutes
		+	# Incubate gel in rinse solution for 30 minutes
		+	# Look for band on illuminator
		+	## If band exists, cut out of gel with razor
		+	# Follow protocol from Promega Wizard miniprep kit
		+	## melt the gel for 5-10' at 70°C
		+	## add 1mL purification resin and mix
		+	## put the resin in the column
		+	## wash with 2mL 80% isopropanol
		+	## spin 2' at 14000 rpm
		+	## air dry for 5'
		+	## add 50uL autoclaved water and incubate 2'
		+	## spin 30 seconds at 14000 rpm
		+	# Lyophilize and resuspend in 20μL water
		+	# perform second cut on vector in a 50μL mix
		+	## Use 20μL from previous gel purification
		+	## Add 5μL BamHI buffer
		+	## Add 24μL water
		+	## Add 1μL BamHI
		+	## Incubate at 37°C overnight
		+	# Run a 1% Gel
		+	## (25 mL TBE, 0.25g agarose)
		+	# Incubate gel in ethidium bromide solution for 30 minutes
		+	# Incubate gel in rinse solution for 30 minutes
		+	# Look for band on illuminator
		+	## If band exists, cut out of gel with razor
		+	# Follow protocol from Promega Wizard miniprep kit
		+	## melt the gel for 5-10' at 70°C
		+	## add 1mL purification resin and mix
		+	## put the resin in the column
		+	## wash with 2mL 80% isopropanol
		+	## spin 2' at 14000 rpm
		+	## air dry for 5'
		+	## add 50uL autoclaved water and incubate 2'
		+	## spin 30 seconds at 14000 rpm
		+
		+	===Ligation===
		+	This also runs overnight
		+	# Premix vector insert and Mg2+
		+	## 1μL cut vector
		+	## 5μL insert
		+	## 1μL Mg2+ (20x)
		+	## 5μL H2O
		+	# Incubate mix for 5' at 37°C
		+	# Add 2μL 10x ATP/DTT (10mM/100mM)
		+	# Add 1μL T4 DNA ligase
		+	# Incubate 2 hours at RT and then ON at 16°C
		+
		+	===Transform===
		+	Protocol uses electrocompetent cells. May have to see about using these

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Matt Hartings: Autocreate 2013/01/04 Entry for User:Matt_Hartings/Notebook/Photosynthesis

2013-01-04T14:18:28Z

Autocreate 2013/01/04 Entry for User:Matt_Hartings/Notebook/Photosynthesis

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{|{{table}} width="800"
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|style="background-color: #EEE"|[[Image:Hartings_AU_Photosynthesis_Lab_Header.png|128px]]<span style="font-size:22px;"> Protein Re-engineering</span>
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==Objective==
Learn how to maintain an OpenWetWare Notebook.


|}

__NOTOC__

User:Matt Hartings/Notebook/Photosynthesis/2013/01/04 - Revision history

Matt Hartings at 16:19, 4 January 2013

Matt Hartings at 16:18, 4 January 2013

Matt Hartings at 15:57, 4 January 2013

Matt Hartings: Autocreate 2013/01/04 Entry for User:Matt_Hartings/Notebook/Photosynthesis