Molecular Biology

Genome Modification

Product lists for commonly used protocols from Cold Spring Harbor Protocols.

Kazuaki Ohtsubo and Jamey D. Marth
This protocol was adapted from "Conditional Mutagenesis of the Genome Using Site-Specific DNA Recombination," Chapter 60, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007.

We describe here a procedure for introducing loxP sites into the mammalian genome. A typical gene-targeting approach to create a conditional null allele is presented, in which the initial placement of loxP sites is not deleterious to allele function. This can be modified to include knock-ins of point mutations, with such mutations flanked by loxP sites that can then be recombined by Cre expression. The choice of sequence or regulatory element to modify is dependent on the experimental design. Consideration must be given to the possible production of truncated and altered gene sequence products, or otherwise aberrantly functioning alleles.
Gene Transfer: Delivery and Expression of DNA and RNA Book Cover

Products Available for this Protocol

Protocol Material Description Product #  Product Name Add to Cart
Dimethylsulfoxide D2650 Dimethyl sulfoxide, liquid (temperature dependent), ≥99.7%, Hybri-Max™, sterile-filtered, hybridoma tested Shopping Cart
Dulbecco’s modified Eagle’s medium (DMEM) D7706 Dulbecco’s Modified Eagle’s Medium - low glucose, With 1000 mg/L glucose, L-glutamine, and sodium bicarbonate, liquid, sterile-filtered, cell culture tested
Fetal bovine serum (FBS) F2442 Fetal Bovine Serum, USA origin, sterile-filtered, cell culture tested, hybridoma tested
MEM nonessential amino acids M7145 MEM Non-essential Amino Acid Solution (100×) , liquid, without
L-glutamine, sterile-filtered, cell culture tested
Sodium pyruvate P5280 Sodium pyruvate, cell culture tested, insect cell culture tested,
≥99%, powder
β-mercaptoethanol M3148 2-Mercaptoethanol, for molecular biology, ≥98% (GC/titration)
10,000 units/ml penicillin:10,000 units/ml streptomycin:200 mM L-glutamine G6784 L-Glutamine-Penicillin-Streptomycin Solution Stabilized, sterile-filtered, cell culture tested
NaCl S3014 Sodium chloride, for molecular biology, ≥98% (titration)
Leukemia Inhibitory Factor (LIF) L5158 Leukemia Inhibitory Factor from mouse, recombinant, expressed in Escherichia coli, 10 µg/ml, buffered aqueous solution, cell culture tested
Ethanol E7148 Ethanol, for molecular biology, 190 proof
G418 A8601 G 418 disulfate salt, Biotechnology Performance Certified
Gelatin (Type A, from porcine skin) G2500 Gelatin from porcine skin, Type A
Mineral oil M8410 Mineral oil, mouse embryo tested, light oil (neat)
Oligonucleotides — Innovative line of custom oligonucleotides for your research
PBS P5368 Phosphate buffered saline, pH 7.4, Biotechnology Performance Certified
Proteinase K P6556 Proteinase K from Tritirachium album, lyophilized powder, ≥30 units/mg protein
Restriction Endonucleases and Buffers
EDTA E5134 Ethylenediaminetetraacetic acid disodium salt dihydrate, for molecular biology, ≥99%
Sarkosyl L5000 N-Lauroylsarcosine, neat, ≥95%
Tris-Cl T5941 Trizma® hydrochloride, for molecular biology, Biotechnology Performance Certified, cell culture tested, ≥99.0% (titration)
Trypsin-EDTA T3924 Trypsin-EDTA solution, 1 ×, 0.5 g porcine trypsin and 0.2 g EDTA • 4Na per liter of Hanks' Balanced Salt Solution with phenol red., sterile-filtered, cell culture tested

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Product Association Disclaimer: The products listed for this specific protocol were selected either to match or to supplement the products listed within the actual protocol. The products/reagents have been qualified for usage, but may not have been validated for this specific application. Please refer to the detailed product description on the usage of specific products of interest.