Natural History Museum Protocol for EST sequencing from lamb

 (1) Core out a single, well separated plaque from the agar plate using a 200µl pipette tip and place it into 500µl of SM buffer in a 1.5ml microtube:

SM buffer

5.8g of NaCl

2.0g of MgSO₄.7H2O

50ml of 1M Tris HCl (pH 7.5)

5ml of 2% Gelatin solution

water to 1l

We believe that it is best to use fresh plaques (i. e. 1-2 days after plating) as the plaques tend to diffuse over time and we get the feeling that older plaques give less reliable results

(2) Vortex the plaque, then leave for 2 hours at room temperature or overnight at 4℃to allow phage particles to diffuse from the agar. After this, the phage suspension can be used as a template for PCR (see step (3)) or frozen for later examination. To freeze, add 1 drop of chloroform and 35µl DMSO (i. e. 7% final) and place at -70℃(no need to snap freeze). (N. b. store chloroform with some anhydrous sodium bicarbonate in the bottom of the bottle, about 1 teaspoon per 100mls.)

(3) PCR the phage insert as follows:

for a 40µl reaction use:

4µl of x10 PCR buffer (see below)

4.8µl of dNTP mix (see below)

0.25 µl of Perkin Elmer Amplitaq Taq polymerase (equivalent to 1.25 units)

1µl of each primer (see below)

2µl of phage suspension from step (2)

27µl of water

overlay with mineral oil and cycle through 25 cycles of:

94℃(1 minute)

58℃(1 minute)

72℃(1.5 minutes)

x10 PCR buffer:

670mM Tris (pH8.8)

20mM MgCl₂

dNTP mix: We use the Pharmacia ultrapure deoxyribonucleotides because they are supplied as 100mM solutions at the correct pH. Add 10µl of each deoxyribonucleotide solution to 760µl water to give 800µl of 1.25mM dNTP mix, aliquot and store at -20℃.

PCR primers: We use T3 ( 5¹-AATTAACCCTCACTAAAGGG -3¹) and T7 (5¹-GTAATACGACTCACTATAGGGC-3¹) primers at 20pm/µl.

(4) Run out 8µl of the PCR reaction on a 1% agarose gel to check:

that there is an insert in the clone (if there is no insert, then the amplification product will be about 170 bases long)

size of insert

yield of fragment

(5) Remove oil from the remaining 32µl of the PCR reaction by placing the reaction mixture on a piece of "Parafilm" (or any similar laboratory strech sealing film - NOT clingfilm!) and gently tilting the film. As the drop runs down the tilted surface, the oil sticks to the film and is left behind, the aqueous phase can then be pipetted up and placed in a clean, 1.5ml microtube.

(6) Remove primers and dNTPs from the PCR reaction using Promega's "Wizard PCR preps".

add 100µl of "Direct Purification Buffer" (see below) to the oil-free PCR reaction (32µl) into a 1.5ml microtube, vortex briefly to mix and then add 1ml of "Wizard Resin" and vortex briefly, 3 times over a 1 minute period

remove the plunger from a 3ml, luer l℃k syringe and attach the barrel to a clean "Wizard Minicolumn" (it is important to use a luer-l℃k syringe, rather than a standard syringe to prevent the column blowing off from the syringe), transfer the PCR /resin mix to the syringe barrel, SLOWLY insert the syringe plunger and GENTLY push the PCR /resin mix into the column (discard any fluid that comes through)

detach the syringe from the column, remove the plunger, reattach the barrel to the column, add 2ml of 80% isopropanol, reinsert the plunger and GENTLY push the isopropanol through the column to wash the resin (discard the fluid that comes through)

detach the syringe and place the column in a clean 1.5ml microtube. Spin for 30 seconds in microfuge at full speed (the kit prot℃ol says to spin at 12,000G for 20 seconds, but the speed is probably not crucial as this step is only to dry the resin)

transfer the column to a clean 1.5ml microtube, apply 50µl of water to the column and leave to soak into the resin for 3 minutes. Then spin at full speed for 30 seconds to collect the eluted DNA in the tube - (if the agarose gel in step 4 only shows a weak band, use less than 50µl of water to elute).

Direct Purification Buffer:

50mM KCl (=0.37g/100ml)

10mM Tris-HCL (pH 8.8 at 25℃) (=0.12g/100ml)

1.5mM MgCl₂ (=0.03g/100ml)

0.1% Triton X-100 (=100µl/100ml)

You can buy kits with all the components in, or purchase the resin, buffer and columns separately - we make up the buffer ourselves).

(7) Cycle sequence the purified PCR product.

For Perkin Elmer's "ABI PRISM, Dye Terminator Cycle Sequencing, Ready Reaction Kit with AmpliTaq DNA Polymerase, FS", use:

8µl of purified PCR product from step (6)

8µl of cycle sequencing reaction mix from kit (contains buffer, enzyme, dNTPs, fluorescent ddNTPs, all ready mixed together)

1µl of primer (T3 primer, sequence as above, at 5pm/µl)

3µl of water

We do the reactions on a Perkin Elmer "480 thermal cycler" under the following cycling conditions

rapid thermal ramp to 96℃

96℃for 30 seconds

rapid thermal ramp to 50℃

50℃for 15 seconds

rapid thermal ramp to 60℃

60℃for 4 minutes

repeat this cycle 25 times, then rapid thermal ramp to 4℃and hold at 4℃as required.

n. b.

you probably can get away with <8µl of kit reaction mix (6µl, maybe less, depending on the signal strength that you are seeing on the ABI - if you wish to do this, reduce all the other reagents in proportion.

if your PCR product is really weak on the gel (step (4)), you can use up to 11µl of it, in this case, adjust the water accordingly to keep the final volume constant at 20µl.

(8) Remove mineral oil from the reaction (see step (5))

(9) Remove excess dye-terminators by ethanol precipitation:

place 2µl of 3M sodium acetate (pH 4.6) (3M sodium acetate (pH 5.2) or 3M potassium acetate (pH5.6) may also be used) and 50µl of cold ethanol in a clean microfuge tube

add the oil-free cycle sequencing reaction from step (8) and place on ice or at 4℃for 10 minutes

spin at 14-17,000G for 10-15 minutes

discard supernatant and wash pellet with 250µl of 80% ethanol (n. b. the Perkin Elmer prot℃ol says to use 70% ethanol)

respin for 5 minutes

discard supernatant and dry pellet at 37℃(or under vacuum, if prefered) (n. b. if you have sucessfuly removed the dye terminators, the pellet will probably be invisible.)

store pellet at -20℃(or -70℃) until ready to run

when ready to run, resuspend pellet in 4µl of formamide / EDTA mix (see below) and heat at 90℃for 2 minutes

formamide / EDTA mix:

180µl of deionised formamide

36µl of 50mM EDTA

This is sufficient for 36 samples, prepare the mix fresh from the 2 stocks immediately prior to use.

Our standard gel run conditions are either:

2500V, 24W, 40mA, 10 hours using 9 parts Sequagel-6:1 part Sequagel-XR (Naional Diagnostics) or 2500V, 30W, 40mA, 14 hours using 19:1 Acrylamide-Bis Acrylamide (Amresco)