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					Lab Protocol MPIMG, Dept. Lehrach, AG Janitz

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Spotting of PCR-Membranes (”wet”)

Materials
 Filter paper, Gel blotting paper (Schleicher&Schuell) Filterpapiere als Unterlage und zur ”Befeuchtung" der Membranen mit Denaturierungslösung  0,3% H2O2 to sterilize the sterilization bath and the brush  0,4 M NaOH to denature the PCR products (do not store longer than 4 weeks, do not store in glas but in PE flasks)  70% ETOH to clean the work disk of the robot  30% ETOH / 5 mM EDTA as solution for the sterilizing bath (to clean the pins)  Hybond N+ Membranen (Amersham,UK)  384-well microtiterplates (MTP) von PE Applied Biosystems oder Genetix  384 pin spotting gadget ”Back Plate” (384 pins with 24 rows each 16 pins and a distance of the pins of 4.500 m and a pin diameter at apex 250m.  Salmonsperm DNA [600 ng/l]

Filter paper (Schleicher & Schuell) Gel-Blotting-Papier Format [mm] GB 004 224 x 224 Tickness [mm] 1,2 weight [g/m2] 700 25 426983 quantity Order number

Nylon membranes (Amersham, UK) Membrane Hybond-N+ (22.2 × 22.2 cm) quantity 10 Order number RPN2222B

384-well Microtiterplatns MicroAmp PE Applied Biosystems (MTP) Polypropylen MTP MicroAmp® 384-well Mikrotiterplates quantity 50 Order number 4305505

Salmonsperm DNA (Sigma-Aldrich - Chemie GmbH) SS-DNA Deoxyribonucleic acid Sodium Salt quantity 5g Order number D1626

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Lab Protocol MPIMG, Dept. Lehrach, AG Janitz

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Basics

The PCR products are spotted from special 384-well spotting plates (Genetix) or when using 384well PCR plates (ABT) and the 384-well Peltier Cycler (MJ Research) directly from these plates on 22x22 cm nylon filter (Amersham). The Spotting takes place with the help of a Spotting robot (Genetix or linear drives). The nylon filters are placed on GB004 Gel Blotting Paper

(Schleicher&Schuell), soaked with 0,4 M NaOH solution. The transfer of the PCR products takes place via multiple Spotting (10 times on each position) with a 384 pinhead. The needles have a circular flat point with a diameter of 250 mm. In a Spotting passage of the duration of approx. 12h in each case 5 filter copies are made. On each 22x22 cm nylon membrane 27648 different PCR products (72.384-well mikrotiterplates) as duplicates are spotted. The spots are arranged in 2.304 blocks per 24 Spots. Into the center of every of these 5x5 blocks genomic Salmonsperm DNA (SSDNA) of a concentration of 600 ng/ml is spotted by multiple spotting (10 times) per spot. This spots of high-complex DNA results in signals in each individual oligonucleotide hybridization and are used as guide dots for image analysis.

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Preparation of the nylon membranes

The Gel Blotting Paper -soaked in denaturing solution (0.4 M NaOH)- is placed on a polystyrene disk (base of the membranes, Slab) without air bubbles and free from creases. Nylon membranes (Hybond N+, Amersham) soaked in 0.4 M NaOH were placed on this soaked Gel Blotting Paper. Bubbles are removed by rolling a glass pipette.

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preparation of the robot

The working desk of the robot has to be cleaned with 70% ethanol before each run. The sterilization bath (with appropriate brush) must be sterilized before use for 20 minutes with a solution of 0.3% H2Ò2. After rinsing twice with A. bidest. the sterilizing bath is fastened in the apprpriate support on the working support and filled with 30% ethanol / 5mM EDTA. For cleanig the 384 pin "single spring" or "back plate" spotting gadget with 250 µm pins has to be separated into its component parts. The pins and the plate are cleaned in the ultrasonic bath with technical ETOH, after drying the gadget is assembled again, sterilized by UV irradiation and built into the robot arm.

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Lab Protocol MPIMG, Dept. Lehrach, AG Janitz

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The microtiterplates (MTP) with the PCR products are carefully placed into the hotel. Each used microtiterplaes must be provided with a bar code readable for the robot, which is placed at the "narrow side" the MTP.

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Spotting ("wet")

After loading of the desired spotting program the robot is started. It takes a MTP from the hotel and places these into the plateholder. The cover of the MTP is removed, the bar code read and the needles of the gadgets dips for 100 ms into the wells of the MTP. The taken solution will be transfered from the needles of the gadgets onto the membranes. After termination of the spottings the membranes are stored until procesing at 4°C in 22.5x22.5 cm plexiglass plates (Genetix), but not longer than 48 hours.

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Filter Processing

The permanent immobilization of the DNA takes place in a processing procedure, which comprises the following steps: 1) Denaturing: put the nylon membrane (do not dive in!) on 0.4 M a NaOH solution for 2 min (change solution after each filter). 2) Washing: dive the nylon membrane into 5x SSC for 2 min (change solution after each filter). 3) Drying: air-dry membrane after putting on filter paper at RT for 60 min. 4) Baking: bake the membrane at 80°C for 30 min. 5) UV: UV cross link using the UV Stratalinkers 2400 (Stratagene) program: AUTO CROSS LINK. The processed filters are stored up to their further use at -20°C (no temporal limitation).

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posted:11/24/2009
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