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Category Archives: Protocols
Microarray Design
Microarray Design Protocol
Please click on below link from the Vodkin lab for complete protocol.
Microarray Construction and Design
Overview
Microarrays on chemically treated glass slides are made by dispensing a small amount of oligonucleotide or PCR product using a solid dispensing mechanism. Common dispensing mechanisms are tweezers, rings, split pins, inkjets, or microspotting pins. In the most common system, using microspotting pins with small chambers, a small volume (typically between 100 and 300nl) is taken up from 384-well PCR plates (spotting plates) containing less than 10ml per well. There may be one or more of these pins taking up DNA simultaneously; up to 32 pins are possible on many arrayers. When a pin touches the surface of a slide treated with a special coating, a small volume of DNA is printedonto the slide (about 1/3 to 1/2nl). The pins are mounted in a gridded head, 4.5mm apart, at the end of a robotic arm, which in turn is controlled by a software program directing the arm to create a specified grid of spots.
Metagrids and subgrids
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Tagged , construction, design, microarray, protocol, Protocols, tma
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Reverse Transcriptase Labeling
Reverse Transcriptase Labeling Protocol
Please click on below link from Vodkin lab for complete protocol.
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Tagged , cy3, cy5, labeling, protocol, Protocols, reverse transcriptase
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cDNA Array Blocking
cDNA Array Blocking Protocol.
Please click on below link from Vodkin lab for complete protocol.
Purpose: After spotting cDNAs onto glass slides, the slides must be treated to bind the DNAs to the coating and to denature the DNAs so they can hybridize to the fluorescently labeled probe DNA. During the blocking step the array will disappear so be sure that the boundary of the array has been marked with a diamond pin as described in microarray construction. During the process, the exposed amine (NH3+) groups are blocked by covalently linking them to succinic anhydride.
Transferring DNA Suspensions
Transferring DNA Suspensions
Please click on below link from Vodkin lab for complete protocol.
Transferring final DNA suspension from 96 to 384-well plates using Hydra Robot
Set 384 well plates in baggie in fridge so they’ll be cold while pipetting (to reduce evaporation).
Spin down samples in 96-well plates (4000 rpm, 1 min)
Turn on Hydra:
* one switch on ISO Bar power strip
* one of machine for pumps
* one on syringe machine
Hit EMPTY button on syringe unit to remove water from syringes (stored with water in syringes).
Remove white HYDRA trough that has ridges in bottom Hit pump toggle switches unit(Fill 1, Fill 2, Empty) on wash pump to remove air from the lines.
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Tagged , dna, protocol, Protocols, robot, suspensions, transfer, transferring dna suspensions
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Sephadex PCR Products Purification
Sephadex PCR Products Purification Protocol
Please click on below link from Vodkin Lab for complete protocol.
PURIFICATION OF PCR PRODUCTS WITH SEPHADEX
1. Place the sephadex measuring plate (MultiScreen Column Loader) on a clean piece of saran wrap. Pour some sephadex onto the plate (takes about 3.3 g). Scrape the surface with the Multiscreen metal plate with the plastic scraper so that the sephadex will fill all 96 wells. Place Polyfiltronics 96-well filter plate on top of filled measuring place. Hold together very tightly and flip such that the sephadex falls out of the wells of the mesuring plate into the Polyfiltronics wells. Keeping plates lined up, tap the measuring plate with a marker pen so that all the sephadex comes out. Remove the measuring plate and brush off the bottom of the Polyfiltronics plate with a clean brush and place it on top of a clean piece of aluminum foil.
2. Add 300 l of sterile water to each well of Polyfiltronics plates with the Matrix electronic 8-channel pipettor. Cover with a piece of parafilm. Incubate for 4 hours at room temperature. After 4 hours, place it on top of a 96-well plate 500ul microtiter plate (Evergreen from Phenix, catalog #LMP-8001. Secure with rubber bands and spin them down at 750x g for 90 sec.
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Tagged , pcr, pcr products purification, polymerase chain reaction, products, protocol, Protocols, purification, reaction, sephadex
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PCR Primer Preparation
PCR Primer Preparation
Please click on below link from Vodkin lab for complete protocol on PCR Primer Preparation.
Purchase 25 nmoles from the Keck Center Sequencing Lab. Dissolve in 1.25 ml sterile nanopure water (25nmoles/1.25 ml = 20 nmole/ml or 20 mmole/L which is 20 mMolar). Let set on desk for several minutes to ensure primers are well dissolved. Store at -20 C. (Use 1 ml of primer for four plates of PCR. Store the remaining 250 l at -20C).
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Tagged , pcr, polymerase chain reaction, primer, primer preparation, primers, protocol, Protocols, reaction
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