* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Lab 1 - DNA Isolation from Drosophila melanogaster (Fly DNA Mini
List of types of proteins wikipedia , lookup
DNA barcoding wikipedia , lookup
DNA sequencing wikipedia , lookup
Molecular evolution wikipedia , lookup
Comparative genomic hybridization wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
DNA vaccination wikipedia , lookup
Bisulfite sequencing wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Nucleic acid analogue wikipedia , lookup
SNP genotyping wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Non-coding DNA wikipedia , lookup
Community fingerprinting wikipedia , lookup
DNA supercoil wikipedia , lookup
Molecular cloning wikipedia , lookup
Protocol 9 - Ligation of Restriction Enzyme Cut Genomic DNA into Restriction Enzyme Cut Bluescript© Phagemid Buffer rATP Frag DNA Vector DNA stock conc. desired conc. T4 ligase ddH2O XXXXX XXXXX XXXXX XXXXX FINAL VOL. XXXXXXXX XXXXXXXX XXXXXXXX XXXXXXXX volume to add Supplies needed: Equipment needed: - 10X T4 DNA Ligase Buffer - 10 mM rATP - restriction enzyme cut DNA fragments - restriction enzyme cut Bluescript© phagemid - 1.5 ml sterile microcentrifuge tubes (1/student) - ddH2O - microcentrifuge - P-10, 100 micropipettors - rack to hold microfuge tubes Important Notes to Remember: Use a different pipette tip for each component of the reaction so as to NOT contaminate the stock solutions. As usual, it is very important that you employ sterile technique to avoid contamination. Only remove the enzyme from the freezer or from the ice when you are prepared to add it. When any enzyme is off the ice for a prolonged period, it can lose activity over time if it is not on ice or in the freezer. Minimize the time it is unfrozen. If you are using genomic DNA, avoid excessive pipetting in order to avoid sheering which may cause undesired breakage. □ 1. Determine the final volume of the reaction you wish to perform (e.g. 50 μl). Then, based on the stock concentrations, calculate the necessary amount of each component of the reaction, calculating the amount of water needed last. □ 2. Add these components to your 1.5 ml microcentrifuge reaction tube in the following order: □ □ □ □ □ ddH2O 10 X T4 DNA Ligase Buffer rATP's Digested insert DNA fragments Digested Bluescript© phagemids □ 3. Mix these components by gently flicking the tube with your finger. Use the mini-microcentrifuge to quickly spin the contents at about 3000 RPM for about 3 seconds. □ 4. Add the T4 DNA ligase and incubate the reaction at room temperature for the (22°C) appropriate for 2 to 24 hours. .