20.109(S13):DNA cloning (Day4)

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

1. Prepare a clearly labeled eppendorf tube for each cloning reaction.
2. In the following order, combine 3 μL cloning buffer, 2 μL purified DNA, and 1 μL of vector mix.
3. Incubate at room temperature for 5 min, and then move to ice if you are not yet ready to proceed with the next step. Meanwhile, obtain an aliquot of competent cells from the teaching faculty and let them slowly thaw on ice. Label the side of each tube (and also the top if you like).
4. When both cells and reaction are ready, add 1 μL of the reaction to the cells. Do not pipet up-and-down more than once! Instead, gently mix the cells and DNA by twirling the tube in your fingers.
   • Hold the tube near the top, so that the cells at the bottom stay cold.
5. Incubate the mixture on ice for 20 min.
6. Place the tubes in the 42 °C heat block for exactly 45 seconds, and transfer immediately immediately to ice for 2 min more.
7. Add 250 μL of warm LB to each sample; do not pipet to mix.
8. Place the tubes on the nutator in the 37 °C incubator, and rock them for about 1 hr.
9. Pre-warm 3 LB-Amp-Xgal plates in the incubator at least 20 min prior to using them in the next step.

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.

1. 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.
2. 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.
3. 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

For next time

Reagent list

write something here or not accessible to edit