Team:Nevada/Protocols

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Bacterial Glycerol Stocks

1. Put 0.5mL bacterial culture in a sterile eppendorf tube.
2. Add 0.5mL of sterile 80% (v/v) glycerol solution
3. Freeze in liquid nitrogen and add to -80oC freezer
4. To recover, scrape frozen surface of culture with sterile inoculating needle, and then streak onto LB agar plate containing appropriate antibiotics, or inoculate liquid culture containing appropriate antibiotics.

DNA Quantification using NanoDrop

1. Select Nucleic Acids measurement
2. Initialize the NanoDrop spectrophotometer with 2µL of autoclaved H2O and wipe off
3. Blank (calibrate) the NanoDrop spectrophotometer with 2µL of the same elution buffer used during DNA purification and wipe off
4. Measure 1.5µL of DNA sample and record the concentration in ng/µL

Gel Purification of DNA

1. Cut out the DNA fragment from the agarose gel with a razor blade, while minimizing the size of the gel slice
2. Weigh the gel slice and add 3 volumes of Buffer QG to every 1 volume of gel (100mg = 100µL)
3. Dissolve the gel slice using a 60°C heat block
4. Apply the dissolved gel to the QIAquick column and centrifuge at 13,000rpm for 1 minute
5. Discard the flow-through and repeat Step 4 until all sample has passed through the column
6. Add 500µL of Buffer QG to the QIAquick column and centrifuge at 13,000rpm for 1 minute
7. Wash the column with 750µL of Buffer PE and centrifuge at 13,000rpm for 1 minute
8. Discard the flow-through and centrifuge at 13,000rpm for 1 minute to remove residual EtOH
9. Transfer the QIAquick column to a new Eppendorf
10. Add 35µL elution buffer to the center of the column and wait at least 2 minutes
11. Centrifuge at 13,000rpm for 1 minute

PCR Purification of DNA

1. Add 5 volumes of Buffer DB to 1 volume of PCR sample
   • ex: Add 250µL Buffer DB to 50µL PCR sample
2. Apply this mixture to a QIAquick column and centrifuge at 13,000rpm for 1 minute
3. Discard flow-through and repeat Step 2 until all sample has passed through the column
4. Wash column with 750µL Buffer PE and centrifuge at 13,000rpm for 1 minute
5. Discard flow-through and centrifuge at 13,000rpm for 1 minute to remove residual EtOH
6. Transfer QIAquick column to new Eppendorf
7. Apply 50µL elution buffer to center of the column and wait at least 2 minutes
8. Centrifuge at 13,000rpm for 1 minute

Phusion PCR

• Thermocycling conditions:
  

1. Initial Denaturation: 98°C for 30 seconds
   
2. 25-35 cycles:
   

• 98°C for 10 seconds
• 55°C, 60°C, 65°C for 15 seconds
• 72°C for 15 seconds

1. Final Extension: 72°C for 5 minutes
   

Qiagen Miniprep kit

www.qiagen.com/hb/qiaprepminiprep

Gibson Assembly

1. Please note that this protocol has been optimized for the assembly and cloning of one or two fragments into any vector. For the cloning of larger assemblies, we recommend using the standard protocol provided with the Gibson Assembly Master Mix. Gibson Assembly Reaction Setup: Amount per Reaction

PCR Fragment(s)+linearized vector2-10 μl Gibson Assembly Master Mix(2X)10 μl Deionized H2O XX μl Total Volume 20 μl

1. Optimized cloning efficiency requires about 25–100 ng of vector and at least 2-fold excess inserts. Use 5–10X more insert if the size is less than 200 bp. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.

1. Incubate samples in a thermocycler at 50°C for 15 minutes. After incubation, store the samples on ice or at -20°C for subsequent transformation.
2. Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
3. Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.

Transformation

1. 1.Thaw competent cells on ice.
2. 2.Add 2 μl of the chilled assembly product to the competent cells. Mix gently by pipetting up and down or by flicking the tube 4–5 times. Do not vortex.
3. 3.Place the mixture on ice for 30 minutes. Do not mix.
4. 4.Heat shock at 42°C for 30 seconds. Do not mix.
5. 5.Transfer tubes to ice for 2 minutes.
6. 6.Add 950 μl of room-temperature SOC media to the tube.
7. 7.Incubate the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. 8.Warm selection plates to 37°C.
9. 9.Spread 100 μl of the cells onto the selection plates. Use Amp plates for positive control sample.
10. 10.Incubate overnight at 37°C.