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DNA Fragment Purification from Agarose or AcrylamideFor fragments from 200 bp to 10 kb the agarose purification is ideal.For smaller fragments (20 bp to 400 bp)the acrylamide purification is preferred ...

Source: Contributed by Mohammad Reza Abbaszadegan et al. Abstract: Single strand conformational polymorphism (SSCP)is the most widely used PCR-based methods for point mutation detection. The abnormal ...

一、In a microcentrifuge tube prepare a solution of linear DNA (25-50ng) in deionized water or TE buffer (10-35µl). 二、Add: （一）10X ligation buffer 5µl （二）50% PEG 4000 solution (for blunt ends only) 5µl （ ...

1、Oligo design（1）The 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 where you want to insert the ...

The preferred method of de-phosphorylation uses the buffer system described by Pharmacia for most of their restriction endonucleases. You will need:10 x OPA Buffer (100mM Tris.acetate pH 7.5 100mM mag ...

It is best to do a test ligation without insert if the background is low it would not be necessary to screen colonies for insert.1.colony PCR 1) Pick a colony from plate and suspend the colony in 10 µ ...

一.实验目的 1.掌握DNA指纹图谱技术的概念、原理和基本操作过程2.学习DNA的限制性酶切的基本技术3.掌握琼脂糖凝胶电泳的基本操作技术，学习利用琼脂糖凝胶电泳测定DNA片段的长度，并能对实验结果进行分析。二.实验原理1984 年英国莱斯特大学的遗传学家Jefferys及其合作者首次将分离的人源小卫星DNA 用作基因探针，同人体核DNA 的酶切片段杂交，获得了由多个位点上的等位基因组 成的长度不 ...

1.Excise band of interest from a TAE gel; estimate volume by weighing the gel slice.2.Dissolve the gel slice in 3 volumes of NaI (from Geneclean kit)at 55E C for 5 min or until the gel dissolves.3.Add ...

Prepare a ligation mix: ...

1.Crash Individual sand flies in 0.5 ml lysis buffer (50 mM NaCl 10 mM EDTA50 mM Tris-HCl pH 8).2.Incubate The sand fly homogenates with 100 ng/ml Rnase at 37 ℃ for 30 minutes.3.Incubate The cell lysa ...

Subcloning should be easy and fast and work every time. The following protocols minimize the number of manipulations required to prepare DNA fragments for ligations thereby both saving time and increa ...

Instructions Modified for Simpson Lab1.PCR: Perform a standard PCR reaction using taq polymerase. (Do not use tfl as the PCR product must have a 3' adenine overhang that only taq generates.) Include a ...

Linker Ligation (with T4 ) DNA LigaseIn a microcentrifuge tube prepare a solution of blunt ended dephosphorylated DNA (100-500ng) in TE buffer (5-7µl). Add 1-2µg of phosphorylated linkers in 5µl of TE ...

Use from 0.01 - 0.1 gram plant material.Grind the plant material with liq.N2 in a mortar.We normally use some alumina to crush hard tissue.Transfer the ground tissue to a eppendorf tube.Add 1 ml extra ...

The overall sequence of events is:• Titer and plate out phage • Lift plaques onto filters and prepare them for screening • Make a probe • Hybridize the probe to the filters • ...

Preparation of linear polyacrylamide used as carrier in ethanol precipitation of nucleic acidsLinear polyacrylamide can be used as an efficient neutral carrier for precipitating nucleic acids with eth ...

Preparation of Silica1. Suspend 5 g of silica (Sigma S-5631) in 50 ml of PBS.2. Allow the silica to settle for 2h.3. Discard the supernatant containing fine particulate matter.4. Repeat steps 2 and 3. ...

competent cells (DH5alpha at competency of 108 cfu/µl of pUC18). The transformation procedure it self is very similar to a standard CaCl2 transformation. Place 100 µl of TB buffer in a tube on ice. Me ...

Recovery of DNA from LMP (Low Melting Point) Agarose Gel1. Separate DNA fragments through an LMP agarose gel containing ethidium bromide (0.5 microgram/ml). 2. Detect DNA by irradiating the gel with l ...

I tried to clean a restriction product 10kb before ligation by a gel extraction method (Qiagen gel extraction kit). But the concentration was too low to do a ligation. I used to clean restriction prod ...