Genome Modification

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View the protocol, "Genome Modification by Inclusion of loxP Transgene Sequences," at BioSupplynet.com

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. ABSTRACT 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.

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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
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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