. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. B. Figure 2. Results from suppression subtractive hybridization. (A) cDNA synthesis from symbiotic (lanes 1 & 2). free-living (lanes 3 & 4). and control (lane 5) RNA. M = 1-kh ladder. (B) Final PCR products from subtracted samples. Lane 1—symbiotic, subtracted: lane 2—symbiotic unsubtracted; lane 3—free-living sub- tracted; lane 4—free-living unsubtracted; lane 5—control subtracted; lane 6—control unsubtracted. M = l-kb ladder. cDNAs were generated and subtracted by using the PCR-Select cDNA Subtraction kit from Clon
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. B. Figure 2. Results from suppression subtractive hybridization. (A) cDNA synthesis from symbiotic (lanes 1 & 2). free-living (lanes 3 & 4). and control (lane 5) RNA. M = 1-kh ladder. (B) Final PCR products from subtracted samples. Lane 1—symbiotic, subtracted: lane 2—symbiotic unsubtracted; lane 3—free-living sub- tracted; lane 4—free-living unsubtracted; lane 5—control subtracted; lane 6—control unsubtracted. M = l-kb ladder. cDNAs were generated and subtracted by using the PCR-Select cDNA Subtraction kit from Clontech. Total RNA was isolated from the algae in the symbiotic state and the free-living state using either the Ambion RNAEasy kit or the Epicentre MasterPure RNA Purification kit. and mRNA was recovered using the Qiagen Oligotex mRNA kit. In both instances, the RNA from each state was separately reverse transcribed using MMLV-reverse transcriptase (Superscript II) to generate double-stranded cDNAs. Both sets of cDNAs were restriction digested with Rsa\ to generate fragments with blunt ends, and the digested cDNAs from the symbiotic state were divided into two aliquots. Each aliquot was ligated to one of two different adaptors supplied in the kit. The strategy of the forward subtraction is to identify cDNAs up-regulated or unique to the symbiotic state of the alga. In this process, an excess of cDNAs from the free-living state (without ligated adapters) was mixed in two separate reactions with the adaptor-ligated cDNAs from the symbiotic state. The two tubes were separately heated to 95°C to denature the cDNAs, and then each was allowed to hybridize at 68°C for 8 h. This step promotes hybridization between the cDNAs expressed in common in the symbiotic and free-living states. To further enhance the subtraction of common cDNAs, the contents of the two tubes were then mixed together without a second denaturation step, combined with an additional excess of heat-denatur
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
