What should this page have?

• Chronological notes of what your team is doing.
• Brief descriptions of daily important events.
• Pictures of your progress.
• Mention who participated in what task.

Using DNA distribution kit plates: Rehydration of registry DNA from kit plate

Wells in the DNA distribution kit plates contain 2-3 ng of dry DNA. This should be enough DNA for a few transformation, but insufficient for immediate restriction and ligation.

Materials

• DNA distribution kit plates
• Sterile distilled water
• Micropipette
• Sterile micropipette tip

Protocol

1. Locate the well with the DNA part you need
2. Using a micro pipette, draw 10 μL sterile distilled water into a sterile pipette tip
3. Use the pipette tip to puncture a hole through the cover foil and into the well containing DNA you need.
4. Release the water into the well and pipette up and down a few times to rehydrate the DNA you need
5. Store the re-hydrated DNA in the -20C freezer. You can either leave it in the original plate, or transfer it into a separate tube (make sure to label the tube!).
6. The rehydrated DNA can be used immediately to transform competent cells. As little as 1 μL should be enough for one transformation.

Restriction Digest

This protocol is for cutting approximately 500ng of plasmid with two different restriction enzymes. This should provide enough cut DNA for both ligation and agarose gel electrophoresis.

Materials

• Plasmid DNA
• 10x Restriction buffet
• Sterile distilled water
• Restriction Enzymes(EcoR1, Spe1, Xba1, and Pst1)
• Thermocycler
• (0.6 mL) Microcentrufuge tube
• Microcentrifuge
• Ice
• Micropipettes and Sterile pipette tips
• Water bath and 37C

Protocol

1. Thaw all solutions and buffers needed. Homogenize the contents by gently flicking the tube. If possible, collect all liquid at bottom of tube by spinning briefly in the microcentrifuge at maximum speed. Place tubes on ice.
2. Label a sterile 0.6 mL microcentrifuge tube with an identifying label for the particular restriction digestion you’re doing. Make sure that you use a permanent marker and the tube fits inside the heating block of your thermocycler.
3. Pipette the following into the tube in the order listed. It adds up to a total volume of 20 μL
1. 7 μL sterile distilled water
2. 10 μL plasmid DNA
3. 2 μL 10x restriction buffer
4. 0.5 μL Restriction Enzyme #1
5. 0.5 μL Restriction Enzyme #2
4. Mix gently by pipetting up and down a few times. You can bring all the liquid to the bottom of the tube by spinning it briefly in the microcentrifuge at maximum speed.
5. Incubate at 37 °C in water bath or thermocycler for 30 minutes to 1 hour.
6. Incubate at 80 °C for 20 minutes. This will denature the restriction enzymes, which otherwise may interfere with subsequent steps.
7. Store the restriction digests in - 20 °C freezer, or proceed immediately with ligation and/or agarose gel electrophoresis.

Growing bacteria from agar stabs onto plates

This method allow us to grow bacteria colonies on pretri dishes

Materials

• agar stabs with the bacteria you wish to grow
• LB agar plates
• inoculating loop
• Bunsen burner
• incubator 37 °C

Protocol

1. Obtain LB agar plates suitable for the bacteria you wish to grow. If bacteria carry plasmid DNA that makes them resistant to a certain antibiotic, use LB agar plates containing the respective antibiotic. For E. coli with no antibiotic resistance (e.g. NEB 10 beta or DH5 alpha cells), which you would need for making competent cells, use LB agar plates without any antibiotics.
2. Warm up the plates to room temperature, and label them.
3. Locate the sample of live bacteria inside the agar stab. Use an inoculating loop to scoop some of the bacteria and streak them onto a plate in a zig-zag line. Be gentle when drawing the line to avoid breaking the agar.
4. Sterilize the loop, wait for it to cool and streak again starting from the end of the first line. This will spread out the bacteria further.
5. Repeat previous step starting at the end of the second zig-zag line. This will spread out the bacteria even more.
6. Repeat again, as needed.
7. Incubate plates at 37 °C overnight (12 - 14 hours) or at room temperature for 24 hours. Make sure plates are incubated with agar layer on top (and lid on the bottom).
8. Plates with colonies can be stored at 4 °C (fridge) and can be used for at least a couple of weeks. Make sure plates are sealed with parafilm, labeled and oriented correctly (agar on top) while stored in the fridge.

Transformation

A process in which we stress competent cells in a series of times heat shocks and ice baths. This allows the cells to take in the plasmids we want them to have.

Materials

• 100 μL aliquots of competent cells
• Ice block
• DNA ligation mix
• Micropipettes and sterile pipette tips
• Glass beads
• Control plasmid DNA
• Water bath at 42 °C (or 37 °C)
• Luria broth
• Incubator 37 °C with a rotisserie
• Luria Broth agar plates (with desired antibiotic)
• Heat block