|
|
 |
|
|
|
BSGC PROTOCOLS AND METHODS
|
|
|
|
|
|
Here are descriptions of our protocols and methods, sorted
according to the steps in our experimental pipeline.
Cloning:
-
Manual LIC
-
LIC Primer Design
This protocol describes the procedures needed to design the primers for
amplification of the
inserts that will be cloned into the BSGC LIC vectors (pB1-pB7).
[More Information (PDF)]
-
LIC Vector Prep
This protocol describes the standard procedure for preparing
LIC Vector Stocks. Qiagen Kit purified vector, 5 ug, is digested with
SmaI in 50 ul NEB#4. Digestion checked using 1 ul on agarose gel
electrophoresis. T4 DNA polymerase exonuclease digestion in the
presence of dATP yields LIC sticky ends. Gel purification of vector is
not essential if SmaI digestion is complete, however if background is
high, it might be necessary.
[More Information (PDF)]
-
LIC Insert Prep Manual
This protocol describes the standard procedure for preparing LIC
Insert Stocks. Qiagen Kit purified Target PCR, 1 ug, is digested with
T4 DNA polymerase exonuclease digestion in the presence of dTTP yields
LIC sticky ends.
[More Information (PDF)]
-
LIC Transformation Manual
This protocol describes the standard procedure for LIC
reactions of prepared vector and Target Insert and transformation into
competent TOP10 cells. The transformation is split between plating and
liquid culture. The plating gives individual colonies for clone
isolation. The liquid culture yields a "mixed" miniprep of plasmid for
characterizaton of the reaction/transformation and potential isolation
of insert containing vectors.
[More Information (PDF)]
-
T4 Insert Prep Manual
A microplate of normalized clean PCR product is treated to produce
T4-treated inserts.
[More Information (PDF)]
-
Robotic LIC
-
PCR Setup
PCR setup using Biomek 2000.
[More Information (PDF)]
-
PCR Normalization
Input: Cleaned-up PCR product plate and quantitation data.
Output: Normalized plate, 1 picomole in 50 ul water.
Next method: T4 polymerase reaction.
[More Information (PDF)]
-
PCR cleanup
This procedure removes the PCR reagents from the PCR product,
resulting in 100 ul of clean PCR product. The next procedure is
Quantitation.
[More Information (PDF)]
-
Insert Prep
This protocol describes the standard procedure for preparing
LIC Insert Stocks on the Biomek 2000. Purified Target PCR DNA is
normalized and 0.4 pmol DNA is digested in a 40 ul reaction with T4
DNA polymerase exonuclease digestion in the presence of dTTP yields
LIC sticky ends.
[More Information (PDF)]
-
LIC Transform
The LIC reaction inserts the Target gene into the vector
plasmid. During the Transformation reaction, the plasmid is taken up
by an E. coli cell, the cells multiply in the culture media, each
forms a colony when plated on agar prepared with the antibiotic
against which the plasmid confers resistance. See T4 Reaction protocol
and Vector Preparation protocol.
[More Information (PDF)]
-
E-gel Run
This method was developed to analyze the PCR screen of plasmid clones,
with columns of 8 clones per gene. The 10 ul PCR screen rxn is diluted
up to 25 ul in the PCR plate, because 20 ul are needed in each well of
the E-gel. Empty wells must be loaded with 20 ul of water. The DNA
standard is loaded manually. The Biomek loads the samples on the
E-gel on the E-gel Motherbase, which is then plugged in for the
electrophoresis. A U.V.digital picture is taken of the gel and saved
on a disk for transfer to a computer for editing and analysis.
[More Information (PDF)]
Expression:
Cell Paste Preparation:
-
Inoculum for Paste SOP
Inoculum for Maxi Growth of Membrane Proteins.
[More Information (PDF)]
-
Studier New Protocol for Auto Inducing Media
Studier Method for Induction, based on Bill Studier's protocol.
[More Information (PDF)]
Purification:
-
AKTA 3D SOP
Protocol for purifying proteins via the 3DEXPII method
(affinity, desalt, ion exchange) on the AKTA.
[More Information (PDF)]
-
AKTA Ion Exchange (IEX) Chromatography SOP
Protocol for purifying proteins via IEX on the AKTA.
[More Information (PDF)]
-
AKTA Metal Chelating (MC) Chromatography SOP
Protocol for purifying proteins via MC on the AKTA.
[More Information (PDF)]
-
HisTrap HP, Ni Sepharose media
Ni Sepharose media and His Trap HP pre-packed columns utilize a new
ligand chemistry to chelate metal ions for metal affinity
chromatography. This media has a higher binding capacity and tolerates
a wider range of chemical conditions than HiTrap MC media. HisTrap HP
media and columns come pre-charged with Ni. It is recommended to strip
and recharge the media/ columns between use with different
targets. The column can be used up to 6 times with the same target.
[More Information (PDF)]
-
mTEV Digestion
Protocol for cleaving proteins with mTEV protease.
[More Information (PDF)]
Protein Characterization:
-
Native PhastGel Electrophoresis SOP
The purpose of the Native PAGE technique is to determine the
homogeneity of a protein according to its banding pattern on the gel.
[More Information (PDF)]
-
Protein Concentration by UV280
[More Information (PDF)]
-
Western Blot SOP
Protocol to detect whether his tagged protein is present in a protein gel
by transferring the protein bands to a nitrocellulose membrane and
probing by using anti-his antibody.
[More Information (PDF)]
Crystallization:
Crystallography:
-
Data Collection Strategy
This flowchart shows how our crystallographers make decisions
related to data collection.
[More Information (PDF)]
-
Recommended flash-cooling and annealing procedures
This flowchart shows our protocol for flash-cooling and annealing crystals.
[More Information (PDF)]
-
Heavy atom soaking experiment
This flowchart shows our protocol for heavy atom soaking.
[More Information (PDF)]
NMR:
[Back to Top]
|
|
| |
|
|
| |
^
| | |
|
|
|
| |
| | |
|
|
