Ligation - 2 $HindlIland ^EcoRI Small DNA fragment (From...

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.Subcloning: Ligation For our ligations, we will use T4 DNA ligase. It catalyzes the joining of two strands of DNA belween the S'-phosphate and the 3'-hydroryl groups of adjacent nucleotides in either a cohesive-ended or blunt-ended configuration forming phosphodiester bonds. The enzyme has also been shoum to cdalyze the joining of RNA to either a DNA or RNA shand in a duplex moleq.rle but will notjoin single-stranded nucleic acids. In this ste,p of,your experiment you will ligate the inserts to the prepared vec'tor (plasmid). As a negative oontrol you wt! add ligase the prepared vector. IXe negatine control win determine how effectively t[e rrector was cleaved and dephosphorfated. Assemble the fo[owiqg in three 200 Fl PCR trbes: Ilbel the trabes with your group's identification- Do not add tape to the tubes as the tubes need to have direct contact to the thermocycler. 1. 9 plffzO 4ld5xligationbuffer 4 pl digested vector (200 ng) (From'Preparation ofVectofl;
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Unformatted text preview: 2 $HindlIland ^EcoRI Small DNA fragment (From freparation oflnsert) I 1tI ligase 2. 9 F!H2O 4 pl5 x ligationbuffer 4 1rl digested vector (200 ng) (From'?reparation of Vector') 2 pI HindIII and.EcoRI Large DNA fragment (From ?reparation oflnserf) I 1rI ligase 3. ll rrlHzO 4[d5xliguionbuffer 4 pl digested vestor (200 ng) (From *Preparation of Vector') I Fl ligase Tap to mir Place PCR tube into a 1.5 ml microcentrifuge tube and centrifuge for 15 seconds at 5,000 rpm. Incubarc l5-l6oc overnight (in the thermocycler, which in this case will serve as an expensive cooling bloct capable of mainAining a constant ' temperatre) . Store all the molecular weight marters, and your large and small DNA inserts inthe frws. . Empty'waste" container inthe trash caos. . Empty ice brrdcets ard return buckets to the shelf. . Wipe off1our bench with water and paper towels....
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This note was uploaded on 07/13/2008 for the course MCDB 104 taught by Professor Lee during the Summer '08 term at UCLA.

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