Team:Johns Hopkins/Notebook/Protocols

DNA Synthesis

Gel Extraction (QIAquick)

1. Excise DNA fragment from agarose gel with clean, sharp scalpel.
2. Weigh gel slice in colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100mg ~ 100µl)
3. Incubate @ 50C for 10 min.
   • SOLUBILIZE AGAROSE COMPLETELY.
   • To help dissolve gel, vortex tube every 2-3 min during incubation.
4. After the gel has dissolved, check that the color of the mixture is yellow. If not, add 10µl 3M NaOAc. Or just add the NaOAc anyway.
5. Add 1 gel volume of isopropanol to the sample and mix
   • Do only if <500bp or >4kb
6. Place spin column in 2mL collection tube.
7. To bind DNA, apply sample to column. Centrifuge 1 min.
8. Discard flowthrough and place column back in same collection tube.
9. Recommended: Add 0.5mL of Buffer QG to column and centrifuge for 1 min.
10. To wash, add 0.75mL of Buffer PE to column and centrifuge for 1 min.
11. Discard flowthrough and centrifuge for an additional 1 min @ 17900 x g (13000rpm).
12. Place column into a clean 1.5mL microcentrifuge tube.
13. To elute DNA, add 30µL Buffer EB to center of membrane, let column stand for 1 min, then centrifuge for 1 min.

Mini-Prep (QIA)

1. Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube.
2. Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 times.
3. Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.
4. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.
5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
6. Centrifuge for 30-60s. Discard the flow-through.
7. Recommended: Wash the QIAprep spin column by adding 0.5 mL Buffer PB and centrifuging for 30-60s. Discard flow-through.
8. Wash QIAprep spin column by adding 0.5 mL Buffer PB and centrifuging 30-60s.
9. Discard flow-through and centrifuge for an additional minute to remove residual wash bufffer. **IMPORTANT: Residual wash buffer will not be completly removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzyme reactions.**
10. Ordered List ItemPlace QIAprep column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 50 µL Buffer EB (10mM Tris-CL, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

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.

PCR Reactions on Small DNA Pieces

Procedure:

1. Combine the following reagents in one tube:
   • 18uL PCR Platinum Supermix
   • .24uL Forward Primer
   • .24uL Reverse Primer
   • .4uL Target DNA
   • 1.12uL Nuclease Free Water
2. Place tube into Thermal Cycler
3. Program Thermal Cycler with desired Denaturing, Annealing and Extension Temperatures.
   • Example:

    94C for 2 minutes
    94C for 30 seconds (Denaturing)
    55C for 30 seconds (Annealing)
    72C for 1min/kb (Extension)
    Repeat Denaturing, Annealing and Extension at least 24 times.
    Hold at 4C

DNA Digestion

DNA Digestion Mixture:

• 25 μl DNA
• 3 μl Buffer 2
• 1 μl EcoRI-HF
• 1 μl PST1
• 3 μl BSA1

DNA Assay

PCR 1.0% Agarose Gel

1. Add .3 grams Agarose to 30mL TAE Buffer in volumetric flask.
2. Stopper volumetric flask with Kim Wipes.
3. Microwave mixture for 1 minute to allow Agarose to dissolve.
4. Prepare BioRad Gel Machine:
   • Place gel stand.
   • Add both gel blocks.
   • Insert comb.
5. Allow mixture to cool down to ~40C.
6. Use micropipetter to seal edges of gel stand with the mixture.
7. Add 3uL Ethidium Bromide to remaining mixture.
8. Mix well.
9. Pour mixture onto gel stand.
10. Cover with Paper Towels and allow to harden.
11. Prepare PCR Reaction tubes:
    • For every 10uL PCR product add 1uL Blue Juice.
    • Mix well.
12. Once gel hardens remove gel blocks and comb.
    • Caution: Remove comb vertically so as not to damage wells.
13. Fill gel machine with TAE buffer till liquid reaches above gel.
14. Load 5uL DNA Ladder.
15. Load 5-10uL samples.
16. Run at 100 Volts for 20-25 minutes.
17. Turn machine off and transfer gel to imaging machine.
18. Position gel and take picture under UV.