20.109(S13):DNA cloning (Day4)
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Revision as of 18:53, 10 January 2013 by AgiStachowiak (Talk)
Contents
Introduction
background on this cloning system -- vector arms and strain
transformation
Protocols
Part 1: Purify 16S PCR band from gel
Part 2: Ligate purified product to vector arms
Part 3: Transform ligated product into cloning strain
Set up microsporidia PCR on this day?
need to do microsporidia PCR on separate day because the Ta etc. is different --> need to find notes on where I planned to put that. possibly Day 2? primers should have arrived by then. or maybe wait until Day 4? don't want to have them balancing lab steps too soon.
- Begin by carefully labeling each PCR tube that you will use with the date, sample name, and a unique symbol and/or color for quick identification. Filling in the cap tab with your team color will usually suffice. Pre-chill the tubes on a cold block.
- You and your partner can now prepare and share a so-called "master mix," which contains every PCR ingredient except the template and the polymerase. Prepare enough for the number of reactions you need to run, plus an additional 10%. Feel free to use the table below for your calculations.
- When the master mix is not in use, keep it on ice.
- Combine 5 μL of template, 45 μL of master mix, and 1 μL of PfuUltra polymerase in a PCR tube. When everyone's reactions are ready, they will undergo the cycling conditions listed below.
- Add the master mix first, because the template alone may freeze. Then add template and polymerase, and finally (gently!) mix the reaction with a larger pipet.
Reagent | Amount for 1 reaction (μL) | Amount for ? reactions + 10% |
---|---|---|
PfuUltra buffer (10X stock) | 5 | |
Primer mix [TO WHAT EXTENT PREPARED FOR THEM?] | 5 of each (Fx and Rx) | |
dNTPs | 1 | |
10% BSA (100X stock) | 0.5 | |
Water | 28.5 | |
DNA template | 5 | N/A |
Segment | Cycles | Temperature (° C) | Time |
---|---|---|---|
1 | 1 | 95 | 3 min |
2-4 | 40 | 95 | 1 min |
58 | 1.5 min | ||
72 | 2 min | ||
5 | 1 | 72 | 10 min |
6 | 1 | 4 | indefinite |