Template Preparation

Once the yeast inserts have been cloned into the M13 vector, the DNA must be amplified and re-isolated. These two tasks are achieved during the template preparation. Each plaque represents an M13 phage possessing a unique yeast insert. Subsequently, each phage is introduced into another bacterial host and grown overnight. During this growth period the phage/insert DNA is amplified. The phage are subsequently harvested and the single stranded DNA is isolated from the rest of the phage. Finally, the DNA is cleaned and resuspended in TE to be se quenced on the Catalyst 800.

Plaque Picking

1-Add 200 µl of 15% glycerol in TE (1:10) to a 96-well titer plate and begin picking plaques.

When picking plaques, try not to pick plaques that are close to one an other. If you accidentally pick two plaques and place them into the same well, the sequence will be useless .

2-Remove the toothpicks and attach lid with aluminum tape (this tape with stands -70°C well).

Preparation of Overnights

1-Add 50 ml (one bottle) of TB salts to 450 ml (one bottle) of Terrific Broth (TB). Place in 37 °C incubator until temperature equilibrium is achieved.

Terrific Broth Solution

Add the components into 900 ml of dH2O. Mix the solutes and then auto clave for 20 min at 15 lb/sq. in. (liquid cycle)

TB Salt Solution

Mix the components and then make up the difference in volume to 100 ml with dH2O. Sterilize by autoclaving for twenty minutes at 15 lb/sq in. (liq uid cycle)

2-Take 0.5 ml of frozen pre-aliquoted NM522 cells and place it in 4.5 ml of LB broth. Grow this culture on the circular rotor for 5-6 hr. After this period, take 100 µl of the NM522 solution and place it in 9.9 ml of TBS (1:100 dilution). Incubate in the 37 °C shaker for 60 -90 min.

NM522 cells are competent cells that will be subsequently infected by the phage . Monitor the time of the second incubation as 1.5 hr is approx. the period of logarithmic growth when the cells are most susceptible to phage infection.

3-While the NM522 starter culture is being incubated, transfer 50 µ l of the phage/TE solution in the 96-well plates into four 24-well culture plates. The Biomek program Phage.fr will perform this operation.

There is a specific order for transferring the TE/phage solution from the 96-well plates to the four 24-well plates. By convention, the first of the 24-well plates will receive the samples in columns 1-6 from rows A, C, E, and G. Plate two will receive from columns 7-12 from the same rows. Plate three will receive samples from columns 1-6, rows B, D, F, and H., and the fourth plate will receive the last set of twenty four. Refer to diagram.

3-Once the NM522 starter culture has incubated for approx. 1.5 hr, remove a 5 ml aliquot and add it to 500 ml of 37 °C TBS. Swirl to mix evenly.

4-Dispense 1.1 ml of the NM522/TBS solution into every well of the four 24 -

well plates.

5-Place the 24-well plates in the 37 °C shaker and incubate overnight at 250 rpm. To maintain a humid environment, place some hot, wetted paper towels in weigh boats with the 24-well plates and cover the entire set with a plastic bag.

It is important to maintain the humidity to ensure bacterial growth. and prevent desiccation.

Phage Harvest

1-Spin down the 24-well plates at 3000 x g for 10 min.

Protocol Number three has a slow acceleration which will prevent the 24 -well plates from cracking under the strain.

2-Collect 1 ml of the supernatant from the four 24-well plates and place it into a Beckman tall 96-well plate (Matrix A2). Transfer into the deep plate according to the same pattern mentioned above ( refer to Diagram on Page 1 ). Minimize the pellet collected. Place a 96-well cover on the deep plate.

The supernatant contains the phage. The pellet at the bottom is merely cells/cellular debris which is why you want to avoid collecting it. The plates may be stored at 4°C at this point.

3-Centrifuge at 3000 x g for 10 min.

This centrifugation will collect more of the remaining bacterial host cells to the bottom of the plate. It is impossible to remove all of the bacterial cells from the solution, but the a significant percentage will be removed.

4-Transfer 600 µl into another tall 96-well plate (Martix A21).

The plates may be stored at 4 °C at this point.

5-Add 120 µl of a 20% PEG/NaCl solution to each well (Matrix A4).

20% PEG/NaCl

Mix the chemicals and add dH20 up to 2 liters.

6-Cap 96-well plate and invert and shake ~20 times. Incubate at room tem perature for 20 min.

The next series of steps will collect the phage from the supernatant. PEG is a bulky carbon molecule which will facilitate the process.

7-Centrifuge at 3000 x g for 15 min.

8-Aspirate supernatant without removing the pellet. Add 580 µl of PEG wash to each well (Matrix A5) .

Polyethylene Glycol Wash Solution

9-Centrifuge at 3000 x g for 5 min and aspirate supernatant. Record which wells are empty and the approximate size of the pellets.

Ethanol Lysis of Phage

1-Resuspend the phage pellets in 188 µl of TE (Matrix A6).

The plates may be stored at 4 °C at this point

2-Shake for twenty minutes on a shaker platform

This resuspension is important in order for effective lysis of the phage. Use a setting of "7" on our Bellco shaker.

3-Add 12 µl of 5 M NaCl (Matrix A7) .

Once the phage has been lysed the DNA will be in solution. The NaCl will help stabilize the charged DNA and will facilitate its precipitation .

4-Add 500 µl of Ethanol/Butanol solution to each well (Matrix A5).

This step should be done in the hood because butanol is semi -toxic. The ethanol/butanol solution will cause the phage to lyse by dis rupting the intermolecular interactions between the proteins thus causing the protein coat to dissociate.

Ethanol/ Butonal Lysis Solution (5:2)

Beware: Make this solution in the hood as butanol these chemicals are vola tile and butanol is semi-toxic.

5-Cap the plate and mix by inverting five times.

The plates may be stored at 4 °C at this point

6-Centrifuge at 3000 x g for 15 min.

7-Decant the supernatant into the waste container located in the hood. Pat dry on a paper towel.

Invert the Beckman plates gently but quickly in one fluid motion. The pellet will contain the phage/insert DNA.

8-Add 60 µl of TE (Matrix A8) . Mix on a shaker for 20 min.

The plates may be stored at 4 °C at this point

Cleanascite Purification

1-Add 60 µl of Cleanascite to each well. Shake on Bellco shaker for twenty minutes.

2-Spin at 3500 rpm for 10 min.

3-Add 6 µl of 5M NaCl to a cetus 96-well plate (Matrix A11).

4-Transfer 90 µl of supernatant to a 96-well filter plate (Matrix A99).

5-Place the filter plate upon the cetus 96-well plate and centrifuge at 3000 x g for 3 min.

6-Add 200 µl of 100% ethanol(Matrix A200) , cap and mix 10x, spin 10 min, uncap.

The addition of the alcohol to the salt solution will cause the DNA to precipitate out of solution. The plates may be stored at 4°C at this point

7-Centrifuge at 3000 x g for 30 min and decant supernatant.

8-Add 188 µl of 70% EtOH (Matrix A6) .

A 70% EtOH wash will clear the salt from the solution by causing the DNA to precipitate while the salt remains in solution.

9-Centrifuge at 3000g for 5 min. Decant the supernatant.

10-Speedvac plates for 30 min (until dry).

11-Resuspend the pellets in 30 to 60 µ l of TE.

The final volume that the DNA is resuspended in depends upon the size of the pellet following the PEG wash. If the pellet was small at that point, then there will probably be very little DNA and resuspending in a large volume makes further sequencing difficult. If the pellet was of modest size, resuspend in the larger volume. The plates may be stored at 4°C at this point

12-Centrifuge at 3000 x g for 1 min.

Collects all the TE and DNA at the bottom of the plate to maximize the rate of resuspension.

13-Mix the template well, and let it remain at room temperature for 30 min. Meanwhile, prepare a 1% agarose gel.

10 X TAE Buffer

Beware: glacial acetic acid is exteremly volatile and caustic. Take proper precautions.

1% Agarose Gel w/v

Add the agarose and the 1X TAE buffer into a flask and heat (microwave) for 1 minute. Remove the agarose solution. Beware: the flask will be hot. Add 2.0 µl of Ethidium Bromide and pour into the gel box. Beware: Ethidium Bromide is a known mutagen and suspected carcinogen. Use appropriate safety measures when handling. Insert a gel comb into the liquid agarose and let the gel solidify. After the gel has solidified, carefully remove the comb. Lower the sides of the gel box and fill the apparatus with 1X TAE buffer

15-Mix 2 µl from sixteen samples of each template plate with 6 µ l of loading dye and load into the gel. Cover and electrophorese at 220 V for 10 min.

It is possible to run four sets of sixteen on a single gel if the electrophore sis is for a short period of time.

16-Cover the template with aluminum tape and label the top according to the project number, plate number, the date, and your initials. Store at 4 °C over night. Subsequently, store at -20 °C until ready to be sequenced.

17-Photograph the gel with under a UV lamp.

The ethidium bromide will intercalate into the DNA structure. When ex posed to UV, EtBr will fluoresce, allowing one to photograph the bands of DNA. The UV light and the polaroid camera will be found in the darkroom. Take two pictures of the gel, one for the general notebook and one for your records.

Last Updated December 10, 1996
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