Difference between revisions of "20.109(S11):Ligate DNA and transform bacteria (Day4)"

Introduction

Protocols

Part 1: Prepare ligation reactions

You will prepare two ligation reactions, one with both backbone and insert, and one with only backbone. The contents will be:

^New England Biolabs sells 10X Ligation buffer to use with their ligase. It contains ATP so must be kept on ice.

If you cannot meet all of the requirements above given your backbone concentration, it may be okay to use less than 50 ng, or to change the ratio of backbone to insert. Please see the teaching faculty to discuss how to proceed.

1. Assemble the reactions in eppendorf tubes but not in the order listed. Please ask if you are unsure what order to assemble the components.
2. When the ligation mixes are complete, flick the tubes to mix the contents, quick spin them in the microfuge to bring down any droplets, then incubate the reactions at room temperature for at least 1 hour.
   • Even if you will only incubate 1 hour, begin preparing Part 2 (e.g., measure OD₆₀₀ values and aliquot Z-buffer into tubes). You can finish Part 2 during a later incubation or after you have finished the transformation.
   • Alternatively, you can continue the ligation reaction for more than 1 hour until you have finished Part 2. Just keep in mind that there is a later half-hour incubation in Part 3 as well.
   • Finally, you and your partner can split up some of the work if that helps, especially since you have all done β-gal assays before.
3. Place the reactions on the 65 °C heat block for 10 minutes to heat inactivate the enzyme.

Part 2: Transfer function for IPTG → lacZ

1. Follow the protocol from Day 2 for performing β-gal assays.
   • Remember to order your samples from least to most expected activity, especially if one person is working without a partner during the ONPG addition step.

Make chart for how they should fill plates

Part 3: Transform bacteria with ligated DNA

You will perform 4 bacterial transformations, one for each of the ligation mixtures, one negative control, and one transformation with 5 ng of plasmid DNA to assess transformation frequency.

1. Prewarm and dry 4 LB+AMP plates by placing them in the 37°C incubator, media side up with the lids ajar.
2. Get an aliquot of competent cells from one of the teaching faculty. Keep these cells on ice at all times. There should be at least 200 μl of cells in each tube. Aliquot 50 μl of cells into 4 clean eppendorf tubes.
3. Add DNA to each tube of cells as indicated below.
   • Ligation reactions, 5 μL of appropriate mixture to each
   • Positive control, 1 μL of pED-IPTG-INS
   • Negative control, none
4. Flick to mix the contents and leave the tubes on ice for at least 5 minutes.
5. Heat shock the cells at 42°C for 90 seconds exactly and put on ice for two minutes. Use your timer.
6. Move the samples to a rack on your bench then use your P1000 to add 0.5 ml of LB media to each eppendorf tube. Invert each tube to mix.
7. Plate 250 μL of each transformation mix on LB+AMP plates. After dipping the glass spreader in the ethanol jar, you should pass it through the flame of the alcohol burner just long enough to ignite the ethanol. After letting the ethanol burn off, the spreader may still be very hot, and it is advisable to tap it gently on a portion of the agar plate without cells in order to equilibrate it with the agar (if it sizzles, it's way too hot). Once the plates are ready, wrap them together with one piece of colored tape and incubate them in the 37°C incubator overnight. One of the teaching faculty will remove them from the incubator and set up liquid cultures for you to use next time.

For next time

Reagent list