Difference between revisions of "Team:Oxford/Notebook/Week1"
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− | <p class="text">* The final concentrations of the primers were noted as they are needed to determine the annealing temperatures for the primers, which can be done using NEB’s | + | <p class="text">* The final concentrations of the primers were noted as they are needed to determine the annealing temperatures for the primers, which can be done using NEB’s <a href="http://tmcalculator.neb.com/#!/">online tool</a>.<br> |
** Add components in order of decreasing volume for maximum ease-of-pipetting.<br> | ** Add components in order of decreasing volume for maximum ease-of-pipetting.<br> |
Revision as of 13:14, 2 July 2015
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22/06/2015 | Whole Team |
Preparation of Stock Solutions1. gBlocksThe gBlocks ordered from IDT arrived in the form of vials of 200ng solid DNA powder. (refer to BioBricks page for information on DNA sequences) The gBlocks were made into 10ng/µl stock solutions in Milli-Q water for storage:
2. PrimersThe forward and reverse primers ordered from IDT came in 32.4nmol and 34.3nmol of solid respectively. (Sequences: Forward - CTTTTTTGCCGGACTGC; Reverse - ATGATTTCTGGAATTCGC) The primers were made into 100µM stock solutions in Milli-Q water for storage:
Preparation of Reaction Solutions1. gBlocks2µl of each stock solution were diluted in Milli-Q water to achieve final solution volumes of 20µl to make 1ng/µl-1 reaction solutions. 2. Primers2µl of each stock solution were diluted in Milli-Q water to achieve final solution volumes of 20µl to make 10µM reaction solutions. (These solutions are labelled as “Prefix primer” and “suffix primer” in eppendorf tubes in the fridge) Polymerase Chain Reaction Set-upThe protocol for running a PCR using NEB’s Q5 High-Fidelity 2X Master Mix can be found here.
* The final concentrations of the primers were noted as they are needed to determine the annealing temperatures for the primers, which can be done using NEB’s online tool. The reaction mixture tubes were positioned in an Eppendorf Mastercycler nexus X2 and the following PCR program was run:
* DNA denaturation can be performed at 98℃ because of the high thermal stability of the Q5 polymerase |