gene therapy & gene editing
gene therapy & gene editing
shRNA Transfection-PDF
Lentivirus, shRNA plasmid, or siRNA?
Choosing between Lentivirus (somewhat stable), shRNA transfer vector (stable/transient), or siRNA (transient). Perform and exhaustive literature search (pubmed) for citations that have reported RNA/DNA delivery...
gene therapy & gene editing
Engineering BioBrick vectors from BioBrick parts/Restriction digest-PDF
Materials
Restriction enzymes (EcoRI, SpeI, XbaI or PstI) from NEB
Bovine Serum Albumin (BSA)
Deionized, sterile H2O
0.5-1 μg DNA
Sterile 0.6mL plastic tubes
Equipment
...
gene therapy & gene editing
PNK Treatment of DNA Ends-PDF
Introduction
This protocol is used to add a phosphate group to the 5' end of a single or double stranded DNA molecule. Most primers, for...
gene therapy & gene editing
Phosphatase treatment of linearized vector-PDF
To minimize self-ligated vector in your transformation, treat your linearized vector with a phosphatase to remove the 5' phosphates necessary for ligation. This should...
gene therapy & gene editing
Purification of DNA-PDF
Alcohol precipitation
DNA Precipitation
Ethanol precipitation of nucleic acids
Ethanol precipitation of small DNA fragments
Isopropanol Precipitation for PCR Purification
use to concentrate DNA...
gene therapy & gene editing
Round-the-horn site-directed mutagenesis-PDF
Background
I was in graduate school and had to make several codon substitutions at the same position in a cloned gene. I came up with...
gene therapy & gene editing
Site-directed mutagenesis-PDF
General Information
Site-directed mutagenesis can be used to change particular base pairs in a piece of DNA. There are several methods for achieving this. The...
gene therapy & gene editing
Cloning Protocol-PDF
PCR
There are two types of PCR polymerases used in the lab, TAQ and Phusion. Phusion is more accurate, having fewer mutations or errors in...
gene therapy & gene editing
Site-directed mutagenesis/Single site-PDF
Materials
Template plasmid purified from a dam+ E. coli strain
Mutatagenesis primers
PfuTurbo DNA polymerase (nonstrand-displacing) and associated reaction buffer
dNTPs
PCR Thermocycler
Dpn...
gene therapy & gene editing
Site-directed mutagenesis/Multi site-PDF
Materials
Template plasmid purified from a dam+ E. coli strain (not JM110 or SCS110)
Mutagenesis primers (one permutation, each on the same strand)
PfuTurbo...
gene therapy & gene editing
Stitching Genes by PCR-PDF
Introduction
This method allows you to "stitch" genes or coding sequences together when there are no convenient restriction sites at the junction point. It is...