DNA基础知识

Desalting Oligonucleotides by Butanol ExtractionDry the oligonucleotide on the speed-vac prior to butanol extraction. Re-suspend the oligonucleotide in 100 m l of water (HPLC grade or better) and tran ...

Colony Cracking: Quick Test for Inserts in PlasmidsE. coli cells can be disrupted in an alkaline solution containing detergent. The lysate contains enough DNA to be detected in a single lane of an aga ...

Protocols for ET recombination.Oligo designThe 5' end (the homology arm) - choose 42 or more (we usually choose about 50) nts for the homology arms from the target DNA sequence simply according to whe ...

Vector systems for cloning different sizes of DNA fragmentsCell-based DNA cloning has been used widely as a tool for producing quantities of pure DNA for physical characterization and functional studi ...

Vector systems for cloning different sizes of DNA fragmentsCell-based DNA cloning has been used widely as a tool for producing quantities of pure DNA for physical characterization and functional studi ...

Subcloning Tips - very important heating stepSource: SchimmelpenninckAbstract: Put your insert and vector in an Eppendorff tube and then heat it for 5 minutes then add ligation buffer and ligaseAfter ...

The following protocol is designed for subcloning inserts (I) from one vector into another vector (V). The inserts can be anywhere from 30 bp to 8 kb (possibly higher).Perform restriction digests of ...

Construction of homemade 'T-vectors'This method is after Marchuk D. et al. 1991 Nucl. Acids Res. 19(5) pp 1154. You will need: 10 x Taq buffer (Promega)Taq Polymerase (Promega)Phenol/chloroform mix100 ...

Analys of Genomic DNA by Southern Hybridization (Southern Blot) Outline:Localization of particular sequences within genomic DNA is usually accomplished by the transfer techniques described by Southern ...

Analysis of Genomic DNA by Southern HybridizationProbe preparationSelect several independent BAC clones containing the same inserts that will be used as a probe. Confirm the BAC clone integrity using ...

Creating a deletion by PCR splicingContributed by Dr.A.GratchevI didn't want to place this in the methods section due to its simplicity. To delete a desired fragment from existing DNA fragment all you ...

1. Prepare Whatman DE-52 according to manufacturers specifications. Equilibrate at store with 0.02% Sodium azide. 2. Plug a 1 mL (blue) pipet tip with Siliconized glass wool. Add 500 ml of 50:50 DE-52 ...

1. Prepare Whatman DE-52 according to manufacturers specifications. Equilibrate at store with 0.02% Sodium azide. 2. Plug a 1 mL (blue) pipet tip with Siliconized glass wool. Add 500 ml of 50:50 DE-52 ...

Mutagenesis by PCR(adapted from Ito et al Gene 102 67-70)This method needs only a single mutagenic primer (+ 3 other primers which are the same for all constructs if used in the same vector) and woul ...

QuikChange (Stratagene)This is a quick and reliable (and dead easy too!) method for PCR but costs more. It is however highly recommended if you need to make only a small number of mutants or if you ca ...

Preparation of Nested DeletionsProcedure:1) Need to cut 10 µg of plasmid in two spots ('A' and 'B') in polylinker. 'A'cut is 'ith a restriction enzyme which gives a 3' recessed end (exonuclease sensit ...

Generation of uni-directional nested deletions in plasmid DNA1) Digest DNA with two restriction enzymes such that one end is ExoIII susceptible (blunt or 5' overhang) and the other is ExoIII resistant ...

Site Directed Mutagenesis- (Kunkel Method) 1. Phosphorylate mutagenic oligonucleotide. 6 uL Primer 1 uL 20 mM ATP 1 uL 10X T4 Kinase buffer 2 uL T4 Kinase (20 units)Incubate at 37oC for 45 min.2. Sec ...

In vitro Mutagenesis with dut ung single stranded DNASteve Hahnlast modified 3/3/00Kinase Oligonucleotide:(gel purified oligonucleotides give highest mutation frequency)0.5 microgram oligonucleotide2 ...

OverviewTargeted Amplification of Mutant Strand (TAMS) technology allows multiple-site mutagenesis in a simple half-day protocol. It’s the only method that canmutate first strand cDNA directly and pe ...