Team:Johns Hopkins/Notebook/Protocols

Contents 1 DNA Synthesis and Assay Protocols 2 Gel Extraction (QIAquick) 3 Mini-Prep (QIA) 4 PCR Reactions on Small DNA Pieces 5 DNA Digestion 6 PCR 1.0% Agarose Gel 7 QuickChange PCR 8 Making Competent E. coli 9 High Efficiency Yeast Transformation 10 E. coli Transformation 11 Reaction to PCR Beta-Carotene genes out of Cells 12 Synthesis 13 Overlap Extension 14 CPEC

DNA Synthesis and Assay Protocols

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.

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

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.

QuickChange PCR

From Marty Taylor of the Boeke Lab:

Based on two stage protocol from - W. Wang, B. A. Malcolm, Biotechniques 26, 680 (Apr, 1999). This two-stage protocol allows formation of some of a “hybrid” between WT and mutant, reducing competition between primer and its GC

Design primer to span region to be changed. Depending on the degree of change (single point mutation vs large deletion/insertion) I usually have 15-40nt on either side with perfect match. Order primer and its reverse complement. We have had good luck using Ultramers from IDT up to ~150nt long, haven’t tried anything longer. We do not PAGE purify primers for this application.

For each primer, setup a 25uL reaction (for older PCR machines with 0.5mL tubes, double everything and you may want to extend the annealing/extension times). I usually do a number of these at a time from the same template, so I make a master mix containing everything but primers, aliquot, and then add primers.

1. Template dNA (~25ng) [1 uL from miniprep]
2. 12.5pMol primer [1.25uL] of 10uM stock – be sure to use only one primer each!
3. 5x Buffer [5 uL]
4. dNTPs (2.5mM each) [2 uL]
5. Herculase II [0.5 uL]
6. H2O → 25uL

Control reaction – this is your background! Add 50uL master mix (without primers) to PCR tube and put it on the cycler, identical to the other tubes. DpnI digest transform like the others.

PCR Protocol:

1. Stage 1: setup 2 tubes as above – one for primer, one for its complement – run 2 or 3 cycles
2. Stage 2: combine the two reactions into one, mix well, run for 18 more cycles
   • 95C, 5min
   • [95C, 30sec / 55C, 30sec / 72C (1min/kB total vector+insert)] – 2-3x in stage 1, 18x in stage 2
   • 72C, 10min
   • Extension time – with the new faster enzymes, 30s/kB may be enough, but have had better luck with 1min

DpnI digest – add 0.5uL DpnI (10U/uL) to each 50uL reaction, vortex, incubate at 37°C for 1-3hrs (longer is better if time permits). Be sure to DpnI digest the control and transform and plate it equally. If you think the reaction is a hard one, you can extend the Dpn digestion to reduce background further (or add more enzyme).

Agarose Gel – WASTE OF TIME! This technique often fails to produce enough DNA to see on a gel and still works. So, basically, this step tells you nothing, since you are going to do the transformation whether or not the gel shows you something. I would do a positive control on the transformation long before I run a gel here.

PCR Cleanup – Herculase II reaction mix is incompatible with our standard competent cells (made using Mn/Ca), reducing the efficiency of transformation by at least 3 logs (perhaps due to detergent in the buffer). A quick PCR cleanup solves this problem.

Transform 5uL into a 50uL aliquot of competent cells. We make our own competent cells; for very difficult reactions, we have had success with supercompetent cells, but I haven’t used these since switching to PFU-Ultra. After heat shock, I grow the cells for 1hr in 500uL SOC and plate both 50uL and 450uL. Sometimes the 450uL plate is a lawn; sometimes there are only 10-50 colonies.

Screening – if your control plate is clean (no colonies), I recommend sequencing (or otherwise screening – if your design allows) 3 clones. Usually at least 2 are positive, but sometimes only 1. Sometimes odd things happen, such as single nucleotide insertions or deletions, errors in the primer sequence itself, or part of the primer annealing with the wrong region of the template, but these results are rarely in all clones sequenced. If there are a lot of colonies on the control (25-50% of the experimental plates), you can sometimes get lucky. Consider longer DpnI digest or repeating.

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:

Temperature	Time	Cycles
98C	3 min	1
98C	30 sec	15 *
55C	30 sec	15 *
72C	total length(kb) * 15 sec	15 *
72C	10 min	1

• 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.