DNA重组

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 ...

Question ...

Desalting Oligonucleotides by Gel FiltrationGeneralDesalting or buffer exchange on a gel filtration column is dependent on volume not quantity of DNA in solution. Many sizes of gel filtration columns ...

Recommended Storage Conditions Probes should be stored frozen both when lyopholized or in solution. Aliquoting your probe into additional vials after resuspension can help to minimize potential breakd ...

General Design GuidelinesThe following guidelines should be taken into account when designing modified oligonucleotides. Sequence Length - SYNTHEGEN can synthesize oligonucleotides from 5 to 110 bases ...

ROX StandardThe final working concentration of ROX Standard in the PCR buffer is 0.5 µMolar (1X). SYNTHEGEN's ROX Standards are shipped in liquid form at a concentration of 100 µMolar (200X). ...

1. Prior to precipitation dilute DNA solution may be concentrated to allow precipitation in smaller volumes.2. Add an equal volume of 2-Butanol to DNA solution and mix by vortexing. 3. Centrifuge samp ...