20.109(S11):Prepare candidate clones in model cell strain (Day5)

Introduction

Protocols

Part 1: Prepare competent JW3367c cells

make sure to describe strain, vs. XL1-blue, above in intro

1. Pick up one 5 mL tube of BL21(DE3) cells. These cells should be in or close to the mid-log phase of growth, which is indicated by an OD₆₀₀ value of ~ 0.6-1.0 for this strain.
2. Measure the OD₆₀₀ value of a 1:10 dilution of your cells. If the cells are not yet dense enough, return them to the rotary shaker in the incubator. As a rule, your cells should double every 20-30 min.

   

   Aspirate the supernatant, as shown, removing as few cells as possible

3. Once your cells have reached the appropriate growth phase, pour them into eppendorf tubes. Spin down 3 tubes of ~ 1.5 mL each for 1 min at max speed (~16,000 rcf/13,000 rpm), aspirate the supernatants, and resuspend in an equal volume of ice-cold calcium chloride (100 mM). Note: you can balance these tubes in the centrifuge with three-way symmetry.
   • If you are nervous about pouring the liquid, you can use your P1000 to pipet 750 μL into each eppendorf twice. Either way, the eppendorf should be quite full when you try to close the cap. You can wear gloves to keep the bacteria from splashing your skin or you can wash your hands after closing all the caps.
   • You may find it easiest to resuspend the cells in a small volume first (say, 200 μL), then add the remaining volume of CaCl₂ (e.g., in two steps of 650 μL) and invert the tubes to mix.
4. Spin again for 1 min. The resultant pellets may occur as streaks down the side of the eppendorf tube, so be very careful not to disturb the cells when aspirating.
   • This strain usually does not streak much, maintaining a relatively compact pellet.
5. This time, resuspend each pellet in 100 μL of CaCl₂, then pool the cells together in one tube.
6. Incubate on ice for 1 hour. (You might work on parts 2, 4, and 5 of today's protocols now, as well as assemble the materials for part 3.)
7. Meanwhile, label four eppendorfs and pre-chill them on ice. The labels should indicate a (+) transformation control (decide and specify), and your three candidate transformations.

Part 2: DNA extraction (mini-prep)

Qiagen kit for better preps in this low copy # plasmid?

1. Pick up your three candidates cultures, growing in the test tubes labeled with your team colour. Label three eppendorf tubes to reflect your candidates (C1-3).
2. Vortex the bacteria and pour ~1.5 mL of each candidate into the appropriate eppendorf tube.
3. Balance the tubes in the microfuge, and then spin them for one minute.
4. Resuspend the cells in 100 μL of Solution I, changing tips between samples.
5. Prepare Solution II by mixing 250 μL of 2% SDS with 250 μL of 0.4M NaOH in an eppendorf tube. Add 200 μL of Solution II to each sample and invert the tubes five or six times to mix. In some cases the samples may appear to "clear" but don't worry if you don't see a big change.
6. Place the tubes on ice for five minutes.
7. Add 150 μL of Solution III to each tube and immediately vortex the tubes for 10 seconds with your vortex set at the highest setting. White clumps should appear in the solution after you vortex it.
8. Place the tubes in the room temperature microfuge and spin them for 4 minutes.
   • While the tubes are spinning, label another set of eppendorf tubes with the candidate names and your team color.
9. A white pellet should be visible when you remove your tubes from the microfuge. Use your P1000 to transfer 400 μL of each supernatant to the appropriate clean eppendorf tube. It's OK to leave some of the supernatant behind. Avoid transferring any of the white pellet.
10. Add 1000 μL of room temperature 100% ethanol to each new tube. The tubes will be quite full. Close the caps and invert the tubes at least five times to thoroughly mix the contents.
11. Microfuge the samples for 2 minutes. It is important to orient your tubes in the microfuge this time since the pellets from this spin may be barely visible.
12. Remove the supernatants using your P1000, taking care not to disturb the pellet of plasmid DNA that is at the bottom of the tube, and put them in a 15 mL conical waste collection tube. Remove as much of the supernatant as possible, but you do not need to remove every drop since you will be washing the pellet in the next step.
13. Add 500 μL of 70% ethanol to each pellet. You do not need to fully resuspend the pellet, but you might invert or flick the tube a few times. Spin the samples one minute, orienting the tubes in the microfuge so you will know where to find the pellet.
14. Immediately remove the supernatant with your P1000, making sure to keep the tip on the side of the tube that doesn't have your pellet. Remove as much liquid as possible, using your P200 set to 100 μL, to remove the last few droplets and/or to streak them up the side of the tube to promote evaporation.
15. To completely dry the pellets, place your rack in the hood with the caps open for ~ 10 minutes. When the pellets are completely dry, add 50 μL of sterile water to each sample and vortex each tube for 2 X 30 seconds to completely dissolve the pellets. The liquid can be brought back to the bottom of the tubes by spinning them in the microfuge for a few seconds. Store the DNA on ice.

Part 3: Transform JW3367c with mutant DNA

1. Prewarm and dry 4 LB+Amp plates by placing them in the 37°C incubator, media side up with the lids ajar.
2. When your competent cells are ready, aliquot 70 μL of cells per pre-chilled eppendorf.
3. Add 2 μL of the appropriate DNA to each tube.
4. Flick to mix the contents and leave the tubes on ice for at least 5 minutes.
5. Continue to heat shock and transform your cells according to the Day 4 Part X protocol.
6. When you get to the 30 minute incubation step, prepare 3 large glass test tubes each containing 3 mL of LB+Amp, and label them with your team color and sample name. (You can also finish part 5 of the protocol if you have not yet done so.)
   • Safety reminder: After dipping the glass spreader in the ethanol jar, then 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).
7. Once the plates are done, wrap them with 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.

Part 4: Count colonies

When you have a spare moment today, count the colonies that arose on your transformed XL1-Blue plate, as well as on your positive and negative control plates. Please put your colony counts on today's Talk (LINK!) page.

Part 5: Prepare DNA for Evaluation

Sequencing Reactions

As we will discuss in lab today, sequencing reactions require a primer for initiation. Legible readout of the gene typically begins about 40-50 bp downstream of the primer site, and continues for ~1000 bp at most. Thus, multiple primers must be used to fully view genes > 1 Kbp in size. Luckily, we only care about a small fragment of pED-IPTG-YFD, i.e., the part containing P_lux-λ.

The recommended composition of sequencing reactions is 200-500 ng of plasmid DNA and 3.2 pmoles of sequencing primer in a final volume of 12 μL. The miniprep'd plasmid should have about 50 ng of nucleic acid/μL on average.

For each reaction, combine the following reagents in an eppendorf tube:

• X μL of your plasmid DNA candidate
• 18 μL of the primer solution on the teaching bench, which (per 18 μL) contains
  • 5.3 μL of sequencing primer at 1 pmol/μL
  • 12.7 μL sterile water

Mix each solution by pipetting and then transfer 12 μL to an 8-PCR-tube strip. Keep track of which sample is in which tube (A-H), and label your tubes on both the side and the top according to the table below. The teaching faculty will turn in the strips at the Biopolymers Laboratory in the Koch for sequencing.

Group	Label Range	Group	Label Range
Red	1-3	Blue	13-15
Orange	4-6	Pink	16-18
Yellow	7-9	Purple	19-21
Green	10-12

For next time

Your Module 1 report revision is due by 11 AM next time, to the 20109.submit address.

Reagent list

• 100 mM CaCl₂