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PROTOCOLS FOR RECOMBINANT DNA ISOLATION, CLONING, and SEQUENCING
recommended
protocol
"DNA Isolation and Sequencing" (Essential Techniques Series)
by Bruce A. Roe, Judy S. Crabtree and Akbar S. Khan
Published by John Wiley & Sons, ISBN 0-471-97324-0 QP625.N89R64 1996 John Wiley & Sons
This manual is a compilation of many of t ...
Protocols for ET cloning
recommended
protocol
ET cloning method uses homologous recombination for DNA engineering in E. coli and was developed in Stewart lab at European Molecular Biology Laboratory.
This ET cloning protocol describes in detail the cloning procedures using ET recombination technique.
...
Lance's Subcloning procedures
new recommended
protocol
A nice protocol/hints on subcloning with detailed procedure and comments. From Bjorkerman group at CIT. ...
Easy Subcloning
new recommended
protocol
Subcloning should be easy and fast, and work every time. This protocol minimize the number of manipulations required to prepare DNA fragments for ligations, thereby both saving time and increasing reliability. Protocol from Michael Koelle's C. elegans Protocol ...
PCR cloning
new recommended
protocol
PCR cloning protocols and methods review:
Considerations for PCR cloning (Excellent guideline for PCR cloning, Invitrogen)
Including tech hints fo ...
LiAc/SS-DNA/PEG Transformation
protocol
High efficiency tranformation protocol from Gietz lab. ...
Manual Large Scale Plasmid, Cosmid, BAC, PAC, and Fosmid DNA Isolation by a Cleared Lysate Method Fo
top rated
protocol
Protocol from Roe's lab Adapted from the Genome Sequencing Center, Washington Univ. ...
DNA Cloning Protocol
protocol
DNA cloning protocols content:
Preparation of oligo solutions
PCR experiments
Digestion of insert DNA
Digestion and dephosphorylation of vector DNA
Ligation of DNA fragments with sticky ends
Ligation of DNA fragments wi ...
Cloning protocols/tips by Astrid
new
protocol
Protocol/tips from Bjerkman lab at CIT. ...
High throughput recombination cloning in E. coli
protocol
Protocol from Finley's Lab at Wayne state University. ...
Heat Shock Transformation of Chemically Competent Cells
protocol
A short yet useful protocol from Chemicon international. ...
HOW TO CLONE IN THE MORRISEY LAB
protocol
The following is a short manual on how we clone DNA into plasmids. These techniques are not to be taken for granted and if one follows the instructions closely, they should be successful! (from Morrisey lab at University of Pennsylavania). ...
PCR Colony Screening
protocol
PCR screen clony using toothpicks. (Chemicon). ...
RAPD PCR Colony Miniprep
protocol
RAPD PCR Colony Miniprep by Gerard R. Lazo. ...
CLONING FROM BEGINNING TO END
protocol
A general protocol for DNA cloning. (Finkbeiner lab, UCSF) ...
E. coli transformation using electroporation
protocol
Protocol from Martin Stocks Ph.D. at Iclectus Ltd. It is part of the intracellular antibody capture (IAC) protocol.
Electroporation is an efficient method for transforming E. coli with plasmid, especially for library scale production of recombinants. ...
MAXIMUM EFFICIENCY COMPETENT CELLS
protocol
Protocol by Sreelekha Devarayalu in Bornstein Group at University of Washington. ...
Isabel’s Magical Cloning Protocol
new
protocol
Gene cloning protocol from Lahn lab at University of Chicago.
1. Insert amplification.
2. Vector/Insert digestion and Vector dephosphorylation.
3. Ligation.
4. Transformation.
5. Colony screening.
6. Mini-prep positive clones and sequencing. ...
Ligation and Transformation Protocol
new
protocol
Protocol on subcloning ligation and transformation. (Tim Fitzwater, SomaLogic, Inc, posted on U.S. Dept commerce/NOAA/NMFS/NWFSC/Molecular Biology Protocols web site) ...
General Cloning Protocols
protocol
A detailed pdf protocol on general cloning from Fero Lab at Fred Hutchinsin Cancer Center. ...
Cloning DNA for sequencing
protocol
Protocol for cloning DNA for sequencing. (University of Southampton, School of Biological Sciences)
PURIFYING DNA FROM AN AGAR GEL
PHOSPHORYLATING PCR PRODUCTS
LIGATION
PREPARATION OF THE LB-AGAR AND SOC
TRANSFORMATION
PICKING POSITIVE COLONIES
SNAP F ...
An optimized recipe for cloning of the PCR product
protocol
Protocol by Zeki Topcu.
This study compares a number of parameters that are important in the ligation of the polymerase chain reaction-amplified DNA inserts into plasmid vectors and their efficient transformation to bacterial cells. The parameters covered w ...
DNA Blunting Protocol
protocol
Protocol from Alam lab at University of Hawaii at Manoa.
A. Blunting Reaction
B. Dephosphorylation of Vector DNA
C. Ligation Reaction ...
BAC Cloning Vector preparation
new
protocol
A protocol for BAC Cloning Vector pBeloBAC11 preparation. (Sangdun Choi, PhD., Caltech)
A. BAC Vector Preparation (Non-CsCL)
B. BAC Vector Preparation (CsCl)
C. Transformation
D. BAC DNA Miniprep Protocol
E. Colony Filters for Library Screening ...
DNA subcloning technical tips
review
Suggestions and technical tips on ligation reaction, transformation efficiency calculation and control, cloning insert and cloning backbone isolation etc. This DNA cloning guidelines are useful for successful and reproducible DNA subcloning. (PJB's cloning hin ...
General Tips To Make Cloning Easy!
review
A general tips and guidance on subcloning (EZ clone system) from Flemington lab at Tulane University.
Whenever possible, we strongly suggest the use of directional cloning strategies (i.e. cloning into a
vector cut with two different restriction enzymes) ...
Typical Cloning Strategies and Tips
new
review
Some useful tips and strategies for DNA cloning. (From Mullin's lab at UCSF) ...
The Joy of Cloning
site
A listing of the protocols that are generally used in the Schimenti Lab. They are extracted from our lab techniques manual, otherwise known as The Joy of Cloning.
Table of Contents
Purification of DNA
Phenol Extraction
Ethanol Precipitation
Pur ...
Some vector sequence and map
site
This is collection of some useful cloning vector sequences and maps (by Dana Boyd at the Dept. of Microbiology and Molecular Genetics, Harvard Medical School).
Arabinose Promoter Plasmids
pBADphoA - Generates phoA fusions under arabinose promoter contro ...
Subcloning oligos into expression vectors
troubleshooting
I've been trying to make several of constructs with various promoter
elements placed in front of a luciferase reporter gene (in a PGL3-based
vector) in order to test them for inducibility in my system. Unfortunately,
... ...
...
Gene construction advice needed
troubleshooting
I'm looking for advice on a gene construction project. I'm trying to
assemble a 219 bp gene from oligonucleotides. I designed the oligos so
that each overlaps the next by ten complementary bases. The plan then is
to fill the gaps, then ligate. I can g ...
CLONING PROBLEMS
troubleshooting
I am making a contructor with pET vector; the question is:
I ligate the vector with the insert and all looks nice, when I transform
E.Coli and I make a mini-prep ( home mini-prep or kit mini-prep from
quiagen ) the plasmid tha appear is littlest than ...
cDNA library screening with antibody
troubleshooting
Can one use antibodies generated against whole cells to screen cDNA
expression libraries (lambda) for transmembrane proteins? Or does the TM
domain cause problems (e.g. ppt, screwed up folding, etc.)? I would ... ...
...
DNA cloning problem
troubleshooting
We have been trying to clone a human gene in to TA vector. We've got
blue/white selection, have screened the white ones directly by PCR, and
then tried to grow up those with inserts - this is where the problem
starts. On trying to grow up for mini-pre ...
help needed with three fragment ligation
troubleshooting
I have a puzzle with regard to a ligation I am trying to perform. I want to ligate three pieces of DNA together, one of 7.1kb, one of 3.6kb
and one of 1.9kb, to form my complete plasmid for bacterial transformation.All the fragments use different sticky ...
T4 DNA ligase frequently asked questions (FAQ)
new
troubleshooting
This is a FAQ on T4 DNA ligase from New England Biolabs.
Q1: What are some potential problems with the ligation reaction using T4 DNA Ligase that can lead to transformation failure?
Q2: What are some other problems that should be considered when trouble sh ...
ligation reaction cosmid DNA with genomic DNA
hot
troubleshooting
I would like a detailed protocol for the ligation of cosmid DNA with genomic DNA.
I have one but it ask me to set up a reaction containing a 9:1 molar ratio of vector DNA:eukaryotic DNA. I don't know how to figure out the molar concentrations. ... ...
...
Blue / white colonies selection problem
troubleshooting
I am using blue/white selection to choose colonies with my insert.
Unfortunately, my white colonies don't have inserts. What could be causing this. ... ...
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Principle of blue white selection of positive recombinant ...
Cloning big fragments
troubleshooting
We have had a continuous problem in cloning large PCR fragments (5-8kb) into a smaller cloning vector (3-5kb). We have used various restriction enzyme site ends. Does anyone have any helpful hints/tricks for cloning 5-8kb PCR or non-PCR DNA fragments into conv ...
Troubleshooting Guide for Plasmid Subcloning
troubleshooting
This cloning troubleshooting guide troubleshoots following aspects of plasmid subcloning:
1. Few or no colonies on control or test plate.
2. Low colony number and 6 or more mini preps are negative.
3. High numbers of colonies on both the Test and the Cont ...
PCR products cloning
hot
troubleshooting
I'm looking for an efficient way to clone 1.6 and 2 kb PCR products into
pET 20b(+) vector,after trying in vain with cohesive end[EcoR1 ,Hind111]
ligation for months. ...
Cloning long DNA fragments inserts
troubleshooting
I had attempted to clone a 5kb insert (from a genomic library) into
the bluescript vector atleast 3 times (into Kpn I - Sma I sites) with
nosuccess. I am doing this only to sequence my inserts. I do a blue
white screening, none of my white colonies co ...
Plasmid Subcloning Troubleshooting Guide
troubleshooting
Troubleshooting guide on plasmid subcloning from EZ clone system. (EZCLoneSystems). ...
Genomic cloning troubleshooting
troubleshooting
Genomic cloning troubleshooting: Evaluating the success of partial fill-in reactions with the Xho I half-site arms cloning strategy
Gary Kobs
Promega Corporation
Promega's LambdaGEM®-11 and LambdaGEM-12 vectors are designed to take advantage of a cloni ...
Competent cells transformation troubleshooting guide
new
troubleshooting
This is a troubleshooting guide for EB5Alpha Competent Cells but is also useful for other types of competent cells. (EdgeBio inc.) ...
Plasmid subcloning and ligation tips
new
troubleshooting
Frequently asked questions on plasmid subcloning and DNA ligation from Promega. (Promega)
Subcloning and ligation tips on:
1. What parameters may need to be optimized for successful ligation reactions?
2. What controls are recommended to ensure s ...
Cloning vector database
database
Cloning vector database currently contains 386 entries for various vectors with maps and accession ID. (S. Yasuda, Department of Microbial Genetics, National Institute of Genetics, Japan). ...
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11-May-2008 08:21 am