Team:Johns Hopkins/Notebook/DNAAssayProtocol

DNA Synthesis and Assay Protocols Page 2

Making Competent E. coli

(From Pam Meluh)

1. Streak out TOP10 (or other strain) from freezer on **plain LB**(or other appropriate plate).
2. Inoculate 25 ml **LB** in a 250 ml flask; grow at 37°C in the air shaker with good aeration.
3. Next day, subculture O/N into a large volume of LB, diluting **1:100** (e.g. 5 ml O/N into 500ml). Make sure to use a large flask, so that the cultures will be well aerated (e.g. for 500 ml, use at least a 2 liter flask).
4. Incubate with rapid shaking at 37°C for 2-3 hours until the OD₆₀₀ reaches ~0.4. If OD₆₀₀ passes 0.5 start over! (Check OD₆₀₀ after 1.5hr; when OD ~0.2, check OD again after 20 min to see if it's at ~0.4)
5. Chill centrifuge and appropriate rotor to 4°C and prepare cold **0.1M CaCl₂**
6. Put culture on ice for 10 minutes. Transfer to bottles and harvest by centrifugation at 4°C (try 10 min. at 4000 rpm for SL-250 rotor).
7. Loosen cell pellets by vortexing the bottles, gently resuspend each pellet in 10-20ml **0.1 M CaCl₂**, and consolidate cells in one bottle. Use **pre-chilled glass pipets** (or pipet up+down w/ cold CaCl₂) to transfer cells and be very gentle with the cells from now on. NO MORE VORTEXING ONCE CaCl₂ IS ADDED. Keep on ice at all times!
8. Let cells sit packed on ice for several hours (at least two hours). Harvest cells by centrifugation.
9. Gently resuspend cells in 1/20 (relative to the starting culture--e.g. 25 ml for a 500 ml culture) of ice **cold 0.1M CaCl₂ containing 15% glycerol**. You can let the cells sit on ice here for a while as well.
10. Aliquot to sterile microfuge tubes (usually .5 and 1 ml aliquots). Flash freeze in liquid nitrogen and store at -80°C until use.
11. Thaw cells as needed for bacterial transformations.
12. Good TOP10 cells should give at least 3x10⁶ transformants per microgram of super-coiled DNA (i.e. 3000 transformants per nanogram)

High Efficiency Yeast Transformation

1. The day before the transformation, there should be yeast on agar plate as colonies or in liquid culture.
2. Inoculate a colony or a small amount of liquid culture (depending on the OD of the culture; usually 250-500 µL) into 5mL YPD liquid culture
3. Place the liquid culture on a rotating drum at 30^oC overnight (prepare two cultures at a time to balance the drum.
4. On the next day check the OD of the inoculate.
5. Re-inoculate the cultures to a final volume of 20 mL, with OD600 of 0.125
   • Each 20 mL should be good for 2 individual transformations
6. Separate the diluted culture into doubles, and incubate on rotating drum at 30^oC for 3 to 5 hours.
7. Spin the culture at 2000rpm for 5 minutes to pellet the cells. Discard supernatant.
8. Wash by resuspending the pellets in 10 mL of water. Spin again at 2000rpm. Discard supernatant.
9. Wash again by resuspending the pellets in 10mL of 100mM lithium acetate (LiOAc). Spin again at 2000 rpm. Discard supernatant.
10. Resuspend the pellets in the residual LiOAc solution in the culture tubes.
11. In a test tube, mix the following solutions for each transformation, vortex to mix:

^Reagents ^ Volume (µL)| ^50% polyethylene glycol (PEG-3350, Sigma) |240 | ^1 M LiOAc |36 | ^Sheared, heat-denatured herring sperm DNA (10 mg/ml)|20 | ^DNA to be transformed (0.5-1 µg/µL) |14 | ^Total |310 |

1. Add the suspend cells (cells from 10mL of culture from step 5) into the PEG mixture specified in the table above. Vortex to mix
2. Inoculate at 30^oC for 30 minutes.
3. Add 36 µL of DMSO into each preparation.
4. Heat shock the preparations at 42^oC for 15 minutes.
5. Pellet the cells at 2000rpm for one minute. Discard supernatant carefully by pipetting.
6. Resuspend cells in 400µL of 5mM CaCl₂ buffer.
7. Incubate at room temperature for 10 minutes.
8. Plate cells directly onto YPD plates with necessary selection criteria (URA-, etc.). Spread with glass beads.
9. Incubate the plates at 30^oC for 2 days.

E. coli Transformation

1. Make sure heat block is holding at 42^oC.
2. Prepare plasmids sample by dilution by adding 3µL sterile H₂O to 1µL of plasmid. Dilution can be ignored if cells will be plated at diluted concentration, or if the plasmids concentrations are low.
3. Thaw one tube of E. coli competent cells (50µL) on ice. One tube is enough for two transformations.
4. Move 25µL of competent cells into new, chilled 1.5mL Eppendorf tube.
   • Keep the competent cells on ice at all time unless otherwise specified.
5. Add 1µL of plasmids into an aliquot of competent cells. Mix by gently flicking. Do not pipet up and down.
6. Incubate the preparation on ice for 20 minutes.
7. Heat shock the preparations at 42^oC for 45 seconds.
8. Immediately place the preparation on ice for 2 minutes.
9. Add 125µL of LB medium to the preparation and incubate at 30^oC for 1 hour.
10. Resuspend cells by gentle pipetting. The plate 100µL of the preparation on LB plates (with any other necessary selection criterion) and spread by glass beads.
11. Incubate the plates overnight at 37^oC

Reaction to PCR Beta-Carotene genes out of Cells

Reaction volume: 20 µL Reagents

 - dNTP(10mM): 0.4
 - primer A (10µM): 1µL
 - primer B (10µM): 1µL
 - Phusion Polymerase: 0.2 µL
 - H2O: 13.4 µL

TAE Gel:

 - 2µL loading dye
 - 5µL DNA
 - 5µL H2O

tPCR

Templateless primer mix Dilute oligos from 60µM (which is what they are delivered at) by a factor of 10.

Use diluted oligo stock to further dilute oligos by a factor of 20.

Add 10 µL of each oligo into an eppendorf tube.

Outer primer mix Add 25 µL of each the first and last primer to an eppendorf tube. Mix thoroughly.

tPCR Reactions Master Mix:

 - dNTP (10mM): 0.5 µL
 - Phusion Buffer: 5µL
 - Phusion Polymerase: 0.25µL
 - H2O: 14.75µL

Reaction tube:

 - Master Mix: 22.4 µL
 - 2.5µL of templateless primer mix

Reaction conditions:

 - 98ºC, 30 s
 - 98ºC, 10 s
 - 54ºC, 30 s
 - 72ºC, 30 s
 - Repeat steps 2 to 4 50 times
 - 72ºC, 10 min
 - 4ºC, hold

fPCR Dilute 5µL of tPCR product by a factor of 5.

Master Mix:

1. dNTP (10mM): 0.5 µL
2. Phusion Buffer: 5µL
3. Phusion Polymerase: 0.25µL
4. H2O: 12.75µL

Reaction tube:

1. Master mix: 20.5µL
2. Outer primer mix: 2 µL
3. diluted tPCR: 2.5 µL

Reaction conditions:

1. 94ºC, 3 min
2. 55ºC, 30 s
3. 72ºC, 63 s
4. 94ºC, 30 s
5. 55ºC, 30 s
6. 72ºC, 1 min
7. Repeat steps 4 to 6 25 times
8. 72ºC, 3 min
9. 4ºC, hold

Run the products on a gel to confirm correct band size

Steps to Assemble:

1. Overlap extension
2. PCR vector pRS416 with primers to linearize
3. Use CPEC to assemble the piece

Synthesis

Overlap Extension

In order to assemble our Vitamin C genes, we used overlap extension PCR. Oligos of up to 60 bp were ordered from IDT, sequentially, in building blocks of up to 800 bp. The first oligo had a 40 base pair overlap with the next, and so on, until the end of that particular chunk of the gene, called a building block. GDP L-Galactose Phosphatase and GDP Mannose-3,5-Epimerase both are made up of two building blocks, and L-Galactose 1-phosphate phosphatase is composed of only one building block. This process is known as templateless PCR. Following this, the PCR product (which will include both incomplete building blocks and a small amount of final product) is PCR amplified using the first and last oligos. This step is called finishing PCR. These building blocks are then purified using a zymogen DNA purification column and then assembled into the final construct in the vector via a CPEC reaction.

CPEC

(Quan 2009)

1. Measure the DNA concentration of each assembly piece
2. Assay 100ng of the linearized vector backbone and equimolar amounts of the other assembly pieces to a 25ul total volume assembly reaction mi#xture accordingly:
   • 100 ng of vector backbone
   • equimolar ammounts of each assembly piece
   • 5ul 5X HF Phusion Buffer
   • 1ul 10mM dNTPs
   • 0.75ul DMSO
   • 0.5ul 2U/ul Phusion Polymerase
   • H2O to 25ul
3. Perform the assembly reaction in a thermocycler as follows:

• Note: the number of repeated cycles should exceed the number of assembly pieces

  4. Transform 5ul of the assembly reaction into 100ul of competent E. coli and/or run a diagnostic agarose gel to check for successful assembly.