XSCREEN PROBE MANUFACTURE

Production of screening probes from L4 library sequences.

PCR procedure: 20µl reactions

All reagents maintained on ice until reaction tubes are transferred to the PCR block

reagent	volume
x10 Buffer (Mg-free)	2
MgCl2[25mM]	1.2
taq[5000Units/ml]	0.1
dNTP[20mM]	2
primer T7plus[100ng/µl]	0.4
primer SAC[100ng/µl]	0.4
ddH2O	14

For ease and consistency produce a single MASTER MIX solution from these reagents sufficient for each set of PCR samples.

Then add Template DNA in 1µl

DNA Source PCR Quantity

cloned DNA 10pg-10ng

genomic DNA 10-100ng

bacterial colony small smear in PCR tube

bacterial cultures 1µl of 1:50 stock dilution

phage suspension from a single plaque 1-10µl (reduce H2O added to suit)

Mix DNA and MASTER MIX aliquot by gentle pipetting

Too much DNA can inhibit the reaction as can contaminating RNA in genomic DNA samples

PCR Protocol

Program Step	temperature	time (seconds)	# cycles
step 1	94	180	1
step 2	94	15	35
	55	20
	72	180
step 3	72	600	1
	4-10	hold

Run PCR products out on 1.5% agarose gel1 :- 4-10µl/lane in loading buffer2

If product of good quality and expected size proceed with digestion; if not repeat PCR protocol.

Production of multi-probe mix e.g. high-frequency clones

Determine the concentration of DNA in PCR tubes: this can be done easily if a quantified Kb DNA ladder is used

Cleaning of PCR Products

Transfer 200-250ng DNA from each PCR to a microconcentrator column: nominal cutoff for dsDNA ~125bp

Usually make up multi-probe mixes from 10-12 clones/column i.e. ~100-200µl total PCR product

Make volume up to 500µl with ddH2O

spin at 1000g for 5minutes

discard filtrate

Repeat x3

Make up retentate to ~200µl with ddH2O

Digestion: for inserts from EcoR1:Xba-1 cloning site

Make up restriction enzyme mix as follows:

reagent	volume(µl)
x10 Multi-core Buffer	40
ECOR1 [10000U/ml]	2
Xba-1 [10000U/ml]	2
ddH2O	196

Add enzyme solution to washed PCR products

Mix by gentle pipetting

Incubate at 37°C in waterbath for 60-90 minutes with occasional mixing by column inversion

Make volume up to 500µl with ddH2O and add to spin column

spin at 1000g for 5minutes

discard filtrate

Repeat x3

Make up retentate to ~200µl with ddH2O

PCR Product Check

Run PCR products out on 1.5% agarose gel1:-10-20µl/lane in loading buffer

Digestion is confirmed by a shift in fragment size range of Å120bp; if not repeat digestion protocol.

Make 1:1000 dilutions5 of PCR samples used to generate probes: remember to include some controls to check levels of non-specific hybridisation, which may occur if the enzyme digestion was incomplete.

Spot 1µl samples of each diluted PCR product on a Nylon transfer membrane

Bake at 80ûC for 2hr

Transfer membrane to roller bottle and pre-hybridise

Dilute 25ng total probe DNA in 45µl ddH2O

Denature by heating at 99ûC for 5 minutes

Centrifuge briefly

Add denatured DNA to 'rediprime' tube and mix contents by gently flicking the tube

Centrifuge briefly

Add 5µl of[32P]dCTP and mix by gently pipetting contents

Centrifuge briefly

Incubate at 37ûC for 10-15 minutes

Stop reaction by addition of 5µl 0.2M EDTA

Denature labelled DNA by heating at 99ûC for 5 minutes

Usually, 0.1µl labelled DNA/cm2 colony membrane sufficient for overnight hybridisation

Wash membrane x2 SSC/0.1% SDS for 30minutes

x0.1 SSC/0.1% SDS for 45minutes

Adjust probe concentration and wash stringency to produce high contrast positive-background hybridisation

1 TBE[X0.5] 50ml

low-melting point agarose 1.6g

Melt agarose in microwave on medium power until agarose completely dissolved

Allow to cool slightly before addition of ethidium bromide

ethidium bromide[50mg/ml] 0.5 microlitres

pour into pre-levelled gel plate containing suitable combs for samples

2 x10 Loading Buffer

0.5M EDTA (pH8.0) 1ml

Sucrose 3g

Bromophenol blue (1%) 0.5ml

Xylene cyanol (1%) 0.5ml

ddH2O 3ml

3 Spin-X UF-blue 100,000MW(Corning Costar, MA, USA) or similar with Nucleotide cut-off dsDNA ~125bp