Team:USP-Brazil/Protocols

Protocols

Cloning

Plasmid DNA isolation

Extraction of plasmid DNA from E. coli by Alkaline Lysis – Miniprep

1.1) Formation of pellets^*

• Transfer 1.5 ml of E. coli sample to eppendorf.
• Centrifuge at 14000 RPM for one minute at 4°C.
• Discard the supernatant.
• Add the remaining E. coli, centrifuge again and discard the supernatant.
• Resuspend in 200 μL of Solution 1 (P1) and vortex to dilute the entire pellet.

^*: Keep all on ice

1.2) Extraction of DNA

• Add 200 μL of Solution 2 (P2) freshly made.

Solution 2^*:
For 2 mL: 1860 μL of milliQ water, 40 μL of NaOH 10N, and 100 μL of SDS 20%.
For 5 mL: 4650 μL of milliQ water, 100 μL of NaOH 10N, and 250 μL of SDS 20%.
^*: The reagents MUST be added in the order described. Stir after each mix. Do not put ice or you might have to redo if a precipitate (crystal) forms.

• Gently invert 6 times.
• Leave at room temperature for 5 minutes.
• Add 200 μL of Solution 3 (P3), flip and put on ice immediately.
• Centrifuge at 14000 RPM for 20 minutes at 4°C.
• Collect the supernatant (with pipette) without getting any of the precipitate pellet moved into the new eppendorf. Collect 200 μL at a time with the pipette tip to prevent getting any of the pellet.

1.3) Precipitation of DNA

• Add 400 μL of isopropanol. Invert 5 times.
• Connect the speed vaccum.
• Centrifuge at 14000 RPM for 10 minutes at 4°C.
• Discard the supernatant and invert eppendorf over paper towel.

1.4) Dry DNA

• Add 500 μL of ethanol 70%. Invert 5 times.
• Centrifuge at 14000 RPM for 5 minutes at 4°C.
• Discard and speed vacuum for 15 minutes.

1.5) Resuspend the DNA and degrade the RNA

• Resuspend in 30 μL of milliQ water with light finger touches.
• Treat with 5 μL RNAse per sample. Resuspend with light finger touches.
• Leave at 37°C for at least half an hour.
• Place in 20°C freezer for overnight storage.

2) Quantification by Nanodrop

• Connect software.
• Select option “Nucleic Acid.”
• Place 2 μL of milliQ water on each pedestal and clean with paper towel.
• Place 2 μL of milliQ water to calibrate the instrument and press “OK.”
• Carry out the “Blank” using 2 μL of either milliQ water or buffer, depending on what you used to dilute your DNA sample.
• Place 2 μL of sample and click measure.
• Observe the quantification of DNA in ng/μL.
• Also observe the ratio of 260/280, which must be around or greater than 1.7-1.8.

3) Digest to linearize the plasmid

Table 1: Candidate promoters from in Pichia pastoris

Reagents	1 reaction
ECO RI*	1μL
Enzyme Buffer	2μL
DNA	2μL
H2O	15μL
Total	20μL

* place the enzyme last

Solutions P1, P2 and P3

P1)

• 23 ml glucose 20%
• GTE 500 ml
• 10 ml EDTA 0.5 M pH 8,0
• 12.5 ml of Tris HCl 1M pH 7,0
• Water to 500 ml
• Autoclave before using

P2)

• H2O 5580 ul
• NaOH 120 ul
• 300 ul SDS 20%

P3)

• KaOH 3M
• 60 ml KOAC 5M
• 11.5 ml Glacial Acetic Acid
• Water to 100 ml
• 11.5 ml Glacial Acetic Acid
• Autoclave before using

Protocol for Miniprep from Plate (used when there was a large amount of samples)

• Take the plates out of the fridge
• Add 1 µL of media (LB) with ampicillin (100 µg/mL) to each well
• Choose colonies by poking with a sterile toothpick and inoculate (put toothpick in well). Cover the 96-well plate with adhesive cover (the worse version) and poke a hole through cover with a needle.
• Put on a shaker at 37°C at 300 RPM for 22 hours
• [in sterile hood] Prepare the 96-well plate with 80 µL with 50% glycerol
• [in sterile hood] Pipette 80 µL of bacteria and mix with a pipette with the glycerol. Cover the 96-well plate with (better) adhesive cover along with plastic cover. Place glycerol stock in -70°C freezer.
• [on ice] Centrifuge at 4000 RPM for 10 minutes (put RNAse on ice to thaw)
• [on ice] Discard the supernatant and turn plates upside down on paper towel
• [on ice] Add 240 µL of solution 1; seal plates with [worse] cover and resuspend with vortex.
• [on ice] Centrifuge at 4000 RPM for 10 minutes
• [on ice] Prepare solution 2
• [on ice] Discard the supernatant and turn plates upside down on paper towel
• [on ice] Add 80 µL of solution 1 – seal plate and resuspend cells with vortex
• [on ice] Take the U-bottom 96-well plate, and add 5 µL of RNAse (10 mg/mL) per well
• Transfer 60 µL of cells to the U-bottomed plate
• Add 60 µL of solution 2; seal with [better] cover and invert plate 10 times
• Leave on the bench for 10 minutes. Spin for a few seconds. (Turn on and preheat heater to 80°C)
• Add 60 µL of solution 3. Seal with [better] cover and invert 10 times
• Leave on the bench for 10 minutes. Spin for a few seconds.
• Take off cover and place plate in 80°C heater.
• [no adhesive cover] Put on ice for 10 minutes.
• [no adhesive cover] Centrifuge at 4000 RPM for 10 minutes
• [no adhesive cover] Attach V-bottom 96-well plate to plate with filter and secure using masking tape
• Transfer all the sample without the pellet and centrifuge at 4000 RPM for 15 minutes.
• Discard the filter plate and add 110 µL of cold 100% isopropanol
• Cover the plate with [better] cover and invert 10 to 20 times
• Spin, change the seal to another cover and centrifuge for 45 minutes (turn on speed vacuum)
• Discard the supernatant and add 200 µL of cold 70% ethanol
• Centrifuge for 5 minutes at 4000 RPM. Discard supernatant
• Dry the plate for 15 minutes using the speed vacuum
• Resuspend with 40 µL of water

To validate plasmid extraction, do a digest and a run a gel or cover and put in -20°C freezer.