User:Matt Hartings/Notebook/Photosynthesis/2013/01/04: Difference between revisions
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==Objective== | ==Objective== | ||
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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Revision as of 08:57, 4 January 2013
Protein Re-engineering | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
ObjectivePrepping some work for the weeks ahead CloningPCR
Purifying PCR product
Restriction of PCR productIf 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 pQE-80 are: BamHI and HindIII
Restriction of vectorRun the cuts at the end of the day
LigationThis also runs overnight
TransformProtocol uses electrocompetent cells. May have to see about using these |