1,721
edits
Richard Lab:Amplified insert assembly: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Richard Lab:Amplified insert assembly (view source)
Revision as of 15:20, 25 January 2011
, 25 January 2011no edit summary
No edit summary |
No edit summary |
||
| Line 32: | Line 32: | ||
*SpeI Restriction Endonuclease | *SpeI Restriction Endonuclease | ||
*PstI Restriction Endonuclease | *PstI Restriction Endonuclease | ||
*DpnI Restriction Endonuclease | *[http://www.neb.com/nebecomm/products/productR0176.asp DpnI] Restriction Endonuclease | ||
*Vent DNA Polymerase | *[http://www.neb.com/nebecomm/products/productM0254.asp Vent] DNA Polymerase (or another equivalent high fidelity polymerase) | ||
*Antarctic Phosphatase | *[http://www.neb.com/nebecomm/products/productM0289.asp Antarctic Phosphatase] | ||
*T4 Ligase | *T4 Ligase | ||
| Line 41: | Line 41: | ||
#[[miniprep|Miniprep]] both "insert" and "vector" from their respective cultures using [[Miniprep/Qiagen kit|a kit]] or [[Miniprep/GET buffer|this protocol]] (30 mins). | #[[miniprep|Miniprep]] both "insert" and "vector" from their respective cultures using [[Miniprep/Qiagen kit|a kit]] or [[Miniprep/GET buffer|this protocol]] (30 mins). | ||
#PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away). | #PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away). | ||
::*Use a high-fidelity polymerase (e.g. pfu Turbo or | ::*Use a high-fidelity polymerase (e.g. pfu Turbo or Vent). | ||
::*Use the same primers you use for colony PCR (Annealing Temp of 55°C). | ::*Use the same primers you use for colony PCR (Annealing Temp of 55°C). | ||
#[[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours. | #[[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours. | ||
#Purify the PCR product using a kit or [[Ethanol precipitation of nucleic acids|this protocol]]. | #Purify the PCR product using a kit or [[Ethanol precipitation of nucleic acids|this protocol]]. | ||
#[[Richard Lab:Restriction Digest|Digest]] insert for 1 hour (adding | #[[Richard Lab:Restriction Digest|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases). | ||
#Add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done. | #Add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done. | ||
#Kill all reactions by incubating for 20 mins at 80°C. | #Kill all reactions by incubating for 20 mins at 80°C. | ||
| Line 64: | Line 64: | ||
**This is why we use a high-fidelity polymerase | **This is why we use a high-fidelity polymerase | ||
**We're sequencing the constructs anyway so we'd spot any mutations. | **We're sequencing the constructs anyway so we'd spot any mutations. | ||
==Contact== | ==Contact== | ||
