




Study with the several resources on Docsity
Earn points by helping other students or get them with a premium plan
Prepare for your exams
Study with the several resources on Docsity
Earn points to download
Earn points by helping other students or get them with a premium plan
Community
Ask the community for help and clear up your study doubts
Discover the best universities in your country according to Docsity users
Free resources
Download our free guides on studying techniques, anxiety management strategies, and thesis advice from Docsity tutors
Material Type: Lab; Class: CELL AND MOLECULAR BIOLOGY: LECTURE AND LAB; Subject: Biology; University: Ramapo College of New Jersey; Term: Unknown 1989;
Typology: Lab Reports
1 / 8
This page cannot be seen from the preview
Don't miss anything!
We will use the 3DNA Kit from Genisphere to hybridize our cDNAs to the microarrays, glass slides spotted with all the genes found in yeast. Once cDNAs are bound to the slide, we’ll hybridize the fluorescent tags Cy3 and Cy5 using the capture sequences introduced by the special 3DNA primers. To measure changes in the mRNA levels in Yeast during the diauxic shift, we need to have a baseline level of gene activity (i.e. mRNA levels). The initial time point ( hours) with excess glucose in the media is our reference level of the mRNAs. The last step in the previous lab was to mix the reference cDNA (Time Point 1 at 9 Hours growth) with cDNA from the later time points (13, 17 or 21 Hours). The ratio of these two cDNAs represents the changing rna expression levels during the diauxic shift in yeast. The slides are prepared by several wash steps before and after hybridization, care must be taken not to contaminate your cDNA sample or the surface of the microarray. Hybridization is done in two distinct steps in this lab. First the cDNA is bound to the microarray. The cDNAs will bind to complementary sequences (i.e. yeast genes) that have been spotted at a particular physical location on the slide (microarray). The cDNAs are incubated for at least 16 hours @ 50ºC to allow specific binding to the microarray. (This step was completed for you before today’s lab) Today’s lab begins with the second step in the hybridization. This step involves binding the 3DNA capture reagents to the cDNAs on the slide. We are employing two different fluorescent dyes, Cy3 (green) & Cy5 (red), which will hybridize to their respective capture sequences on the cDNAs. The different dyes provide a two channel signal that can be represented as increasing (ie red) or decreasing (ie green) images for analysis. Each 3DNA molecule contains ~375 fluorescent molecules providing a uniformly strong signal. Special care must be used when working with the dyes as they are susceptible to oxidation and photobleaching, which can reduce or destroy the signal intensity.
RNA is extremely sensitive to degradation by RNases. How carefully you handle your samples and transfer solutions will have a huge impact on the quality of your microarray data.
streaking. When done, there should be a small volume of water collected at the very bottom of the tube and the array should be dry.
fluorescent dyes both during and after the hybridization process. Store any unused hybridization buffer containing Anti-Fade Reagent at –20ºC. (Only one group needs to do this as you only need 17 μl per slide.)
Post 3DNA Hybridization Wash
Perform the following steps in the dark to avoid degradation and fading of the fluorescent dyes
surface. Agitation during washing may also help to reduce background due to non-specific binding to the surface of the array. Proceed to Signal Detection.
IMPORTANT: Store the array in the dark until scanned. The fluorescence of the 3DNA reagents, especially Cy5 can diminish rapidly even in ambient light because of oxidation.