內切酶列表：EnzymesGeneratingBluntEnds
Asymmetric sequences are indicated by *.Single letter code:R = G or A; Y = C or T; W = A or T; M = A or C; K = G or T; S = C or G;H = A, C or T;V = A, C or G;B = C, G or T;D = A, G or T;N = G, A, T or C. RecognitionsequenceEnzymeAAT^ATTSspIAGC^GCTEco47IIIAG^CTAluIAGG^CCTEco147IAGT^ACTScaIATTT^AAAT SmiICACGTC(-3/-3)^*BtrICAC^GTGEco72ICACNN^NNGTGOliICAG^CTGPvuIICAYNN^NNRTGMslICCC^GGGS......閱讀全文
內切酶列表：Enzymes－Generating－Blunt－Ends
Asymmetric sequences are indicated by *.Single letter code:R = G or A;?Y = C or T;?W = A or T;?M = A or C;?K = G or T;?S = C or G;H = A, C or T;V = A,
內切酶列表：Enzymes－Generating－Blunt－Ends
Asymmetric sequences are indicated by *.Single letter code:R = G or A;?Y = C or T;?W = A or T;?M = A or C;?K = G or T;?S = C or G;H = A, C or T;V = A,
內切酶列表：Enzymes－Generating－5protruding－Ends
Asymmetric sequences are indicated by *.Single letter code:R = G or A;?Y = C or T;?W = A or T;?M = A or C;?K = G or T;?S = C or G;H = A, C or T;V = A,
內切酶列表：Enzymes－Generating－3protruding－Ends
Asymmetric sequences are indicated by *.Single letter code:R = G or A;?Y = C or T;?W = A or T;?M = A or C;?K = G or T;?S = C or G;H = A, C or T;V = A,
DNA的酶學操作
DNA的酶學操作DNA Modifying Enzymes?(Michael Blaber)Introduction to bacterial restriction/modification system. It provides very useful background knowledge
PCR基本實驗方法（五）
Cloning?PCR?ProductsT-A Cloning Strategy:?Taq and other polymerases seem to have a terminal transferase activity which results in the non-templated ad
PCR基本實驗方法（五）
Cloning?PCR ProductsT-A Cloning Strategy:?Taq and other polymerases seem to have a terminal transferase activity which results in the non-templated ad
Cloning－PCR－products－using－TA－vectors
Cloning PCR products using TA vectorsby Paul N. Hengen, Ph.D.?*Methods and reagents is a unique monthly column that highlights current discussions in
EZBlunt載體克隆介紹
EZ- Blunt載體環形圖譜和多克隆位點?EZ-Blunt載體克隆常見問題分析與處理?
DEPHOSPHORYLATION－OF－LINEARIZED－DNA
DEPHOSPHORYLATION OF LINEARIZED DNAALKALINE PHOSPATASE(CIP) DIGESTIONDigestion of protruding 5'-ends1. Precipitate digested DNA and resuspend in a
Generating－stable－cell－lines－in－HEK293
Generating stable cell lines in HEK293Prior to transfection, it is recommended that you linearize your pcDNA gene construct. Linearizing will decrease
Generating－stable－cell－lines－in－HEK293
Generating stable cell lines in HEK293Prior to transfection, it is recommended that you linearize your pcDNA gene construct. Linearizing will decrease
CIP－Treatment
set up the following reaction:CIP RxnH2O7.8 ml10x cip rxn buffer2.0 mlDNA(e.g; 3 kb vector; 0.2 mg/ml; 2 mg total)10.0 ml(1 u/ml) CIP0.2 mltotal20.0 m
cDNA－Libraries
cDNA LibrariesIsolation of corresponding genetic informationInstead of synthesizing a desired gene, can we used the amino acid information to directly
RACE(rapidamplification－of－cDNA－ends)技術1
RACE技術的簡介cDNA完整序列的獲得對基因結構、蛋白質表達、基因功能的研究至關重要。完整的cDNA 序列可以通過文庫的篩選和末端克隆技術獲得。末端克隆技術是20世紀80年代發展起來的。RACE(rapid-amplification of cDNA ends)是通過PCR進行cDNA末端快速克隆
RACE(rapidamplification－of－cDNA－ends)技術2
具體的實驗步驟cDNA第一條鏈的合成：我們建議進行cDNA合成的對照反應，這樣可以對樣品的 cDNA的合成進行鑒定。加入各種試劑之后，在氣浴中42度保溫一個小時。注意： 在水浴或酒精浴中保溫回減少反應體積，從而降低第一鏈的合成效率。將管放于冰上，以終止第一鏈的合成反應。直接進行第二鏈的合成。cDNA
Selfcircularization－of－Linear－DNA
In a microcentrifuge tube prepare a solution of linear DNA (25-50ng) in deionized water or TE buffer (10-35μl).Add:10X ligation buffer?5μl,50% PEG 400
與II型核酸內切酶有關的幾個概念
粘性末端：cohesive ends是指DNA分子在限制酶的作用之下形成的具有互補堿基的單鏈延伸末端結構，它們能夠通過互補堿基間的配對而重新環化起來。平 末 端 ：Blunt end在識別序列對稱處同時切開DNA分子兩條鏈，產生的平齊末端結構。則不易于重新環化。同裂酶：isoschizomers 能
Cloning－of－small－RNAs－with－5’－phosphate－and－3’－OH－ends2
3’ Adaptor Ligation and PurificationHeat shock the RNA by putting at 90°C for 30 seconds. Snap cool on ice.Set up the 3’Adaptor ligation reaction in a
Cloning－of－small－RNAs－with－5’－phosphate－and－3’－OH－ends3
Load your precipitated PCR samples into 2 consecutive lanes so as not to overload the lanes. For each different sample, I would run a separate ladder
Cloning－of－small－RNAs－with－5’－phosphate－and－3’－OH－ends01
IntroductionThe following protocol describes a procedure for the purification and cloning of miRNAs and other small RNAs in the? 20-30 nucleotide size
PCR
PCRPolymerase Chain Reaction1) Add the following to a microfuge tube:10 ul reaction buffer1 ul 15 uM forward primer1 ul 15 uM reverse primer1 ul templ
酶切反應注意事項
一、 建立一個標準的酶切反應目前大多數研究者遵循一條規則，即10個單位的內切酶可以切割1μg不同來源和純度的DNA。通常，一個50μl的反應體系中，1μl的酶在1X NEBuffer終濃度及相應溫度條件下反應1小時即可降解1μg已純化好的DNA。如果加入更多的酶，則可相應縮短反應時間；如果減少酶的用
DNA標記
DNA標記（主要內容如下）??DNA Labeling by Nick Translation??Random Primed Labeling??End-Labeling??Purification of Labeled DNA??Non-isotopic Labeling??OthersDNA L
PCR的下游應用
·?????????Agarose Gel Electrophoresis of PCR Products?(Robert H. Cruickshank)·?????????Agarose Gel Electrophoresis of PCR Products?(Immunology Resourc
酶切反應的建議
酶切反應建議一、?建立一個標準的酶切反應目前大多數研究者遵循一條規則，即10個單位的內切酶可以切割1μg不同來源和純度的DNA。通常，一個50μl的反應體系中，1μl的酶在1X NEBuffer終濃度及相應溫度條件下反應1小時即可降解1μg已純化好的DNA。如果加入更多的酶，則可相應縮短反應時間；如
PCR的下游應用
??????????Agarose Gel Electrophoresis of?PCR?Products(Robert H. Cruickshank)??????????Agarose Gel Electrophoresis of PCR Products(Immunology Resource)
重組DNA的分離、克隆與測序實驗手冊5
C. Random fragment end-repair, size selection, and phosphorylationSince both sonicated and nebulized DNA fragments usually contain single-stranded end
Observation－of－living－and－plasticembedded－chick－embryos
The development of chick embryos has been studied since Aristotle. It is one of the most intensely studied organisms. One reason for this is that ther
Polymerase－Chain－Reaction－(PCR)－cont.
Polymerase Chain Reaction (PCR) cont.Choice of Polymerases for PCROne of the important advances which allowed development of PCR was the availability