DNA重组

Phenol Chloroform Absolute ethanol 70% ethanol TE buffer (10T/1E) pH 8.0 10% SDS 50 mg/ml lysozyme 5 M NaCl ProcedureMacerate Cyanobacteria in TE Buffer (10T/1E) Pellet the cells by centrifugation for ...

Procedure:1) Use Pharmacia #27-4564-01. With clean flamed scissors and forceps weigh 0.25 g/50 ml conical and add 50 ml/conical of 0.02 M Tris pH 7.6. Allow to dissolve over several days at 4°C.2) ...

PCR-based methods are widely used in plants and animals for marker-assisted breeding and high-resolution mapping. These studies require analysis of large number of samples thus a DNA extraction method ...

Take an oral smear Dissolve in 50 ml H2O through vigorous shaking Pellet cells 5 min at 4000 rpm Re-suspended the pellet in 400 μl Lysis buffer (50 mM TRIS-Cl pH 8 10 mM EDTA 2% SDS) Incubate for 5 mi ...

A nested series of deletions can be produced by: cutting plasmid DNA (2.5ug per time point see below) with an enzyme leaving a blunt (eg EcoRV) or 5'' overhand (eg EcoRI) on the side that the deletion ...

Procedure:1) Need to cut 10 µg of plasmid in two spots (‘A’ and ‘B’) in polylinker. ‘A’ cut is with a restriction enzyme which gives a 3’ recessed end (exonuclease sensitive) next to insert. Since ...

0.25M HCl (1L)20.66mL HCl 979.34mL dH2O *add acid to wateundefined 1M ammonium acetate 0.02M NaOH (1L)77.08g ammonium acetate 0.8g NaOH Fill with dH2O 0.5X SSC 0.1% SDS (1L)25mL 20X SSC 10mL 10% SDS 965mL dH ...

Smith and Summers 1980. Anal. Biochem. 109:123-129.Digest 10-15 µg genomic DNA with desired restriction enzyme overnight at 37oC. Run digest on a 1% TBE agarose gel at 100V until 1st blue dye reaches ...

Genomic DNA Digest10 µl DNA (10 µg) 3 µl 10X Buffer 1-2 µl Restriction Enzyme (40 units) 3 µl 10mM Spermidine-HCl 3 µl 1 mg/ml BSA 10 µl H2O Total volume: 30 µlNote: Use DNA from a standard tail prep ...

SOLUTIONSLysis Buffer 0.1 M Tris pH 8.0 0.2 M NaCl 5 mM EDTA

Principle: Refer to Southern Transfer with Zetabind membrane. The NaOH transfer may be a more efficient transfer method for larger sized fragments. It is the method of choice for pulsed-field gels con ...

I. Preparing worm genomic DNA: requires 1-2 days to seed agarose plates a few days for the worms to grow 1-2 days to prep the DNA1. Seed large agarose plates with HB101. Agarose is preferred over agar ...

MaterialsHot Probe dNTP Mix:25 mM dATP25 mM dTTP25 mM dGTP2.5 mM dCTPHybridization Solution:5X SSC0.5% (w/v) Blocking Reagent0.1% (w/v) N-lauroylsarcosine Na-salt0.02% (w/v) SDS50% FormamideBlot Wash ...

IRS-1 Probe DNA Prep 1. From glycerol stocks (in �80ºC freezer) grow up bacteria. Use amp LB. 2. Use a Qiagen prep to purify the DNA. 3. Perform the following digest: ...

Preface: Digesting lambda DNA (BRL) with a specific restriction enzyme will result in lambda DNA fragments of known size. Three separate lambda digests (BglII BstEII and XhoI) will give 23 fragments w ...

You will require the following: -Tail buffer 50ml 10% SDS 5ml 1M Tris pH 7.50.5ml 0.5M EDTA5ml 5M NaCl 1.5ml DDW38ml -Phenol/chloroform (1:1 mixture) -0.5M EDTA -4M NH4Ac -Absolute EtOH -70% EtOH -T ...

About 1/2 to 1 cm of tail should be cut from properly marked mice (using toe or ear clipping etc) about the age of 2-3 weeks. Place these tails into marked 1.5 ml Eppindorf tubes for processing; they ...

About 1/2 to 1 cm of tail should be cut from properly marked mice (using toe or ear clipping etc) about the age of 2-3 weeks. Place these tails into marked 1.5 ml Eppindorf tubes for processing; they ...

A. Large scale double-stranded DNA isolationThe method used for the isolation of large scale cosmid and plasmid DNA is an unpublished modification (16) of an alkaline lysis procedure (1718) followed b ...

We store our all of our plasmids as bacterial host stocks at -80 stored in 7% DMSO. Such stocks are very long-lived giving robust bacterial growth even after 10-15 years. However occasionally we hav ...