Term
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Definition
| It is complementary DNA. DNA complementary to the RNA..basically a copy of the mRNA. |
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Term
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Definition
| You use reverse transcriptase to make DNA copies of the mRNA. |
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Term
| What is the primer you use for reverse transcriptase in cDNA production? |
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Definition
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Term
| Can cDNA make functional protein? |
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Definition
| Yes, it just has to be transcribed into mRNA and then translated into protein |
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Term
| Why is cDNA better than genomic for doing research on stuff? |
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Definition
| cDNA is like ready for action in terms of protein production. You don't need like intons/enhancers and all that garbage. |
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Term
| How can you copy cDNA and make alot of it? |
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Definition
| You make it double-stranded, and then put it into a plasmid. |
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Term
| Would cDNA include all the genes? |
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Definition
| No, it only includes the ones that are being used in a specific tissue type. |
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Term
| What is Northern blotting used for? |
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Definition
| To detect the size and AMOUNT of a specific mRNA! |
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Term
| How do you do Northern Blotting? |
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Definition
| god this blows. Ok: Use a bead with poly-T to trap the mRNA from a lysed cell (because of the poly-A tail. Then wash away the other stuff. Then use salts to get the mRNA off the beads. Then put the mRNAs into a gel and separate according to size. THEN, transfer to blotting paper and add a radioactive probe to the mRNA you are looking for. Then XRAy it. |
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Term
| What would be an example of how Northern blotting could be useful? |
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Definition
| 1) See which mRNAs are being expressed in which tissues. 2) see how much mRNA is being made at what times--like PEPCK under high/low glucose for example |
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Term
| How could you analyze splicing defects by northern blotting? And what would you see in a homozygote/heterozygote for these defects? |
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Definition
| With a heterozygote, you would see one normal sized band of the mRNA in question and one abnormal. With a homozygote, only one abnormal. |
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Term
| What are some uses of cloned DNA? |
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Definition
| To make alot of a rare protein (eg Tissue Plasminogen Activator), put it into a plasmid and make lots of it. |
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Term
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Definition
| Its basically a Northern blot on crack. Can do up to 20,000 mRNAs at once. |
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Term
| How do you do microarray? |
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Definition
| Put exonic DNA sequences on a chip. Take mRNA and use RT to make cDNA labeled with flourescent nucleotides. Use Red for normal and Green for tumor. Then compare the production by tumor and regular cells as they hybridize to the exonic DNA sequence on the chip. |
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Term
| Does Microarray tell you how much mRNA is being produced? |
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Definition
| No, only whether it is there or not. |
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Term
| If a gene is in the transcriptome, is that all you need to know? ie Can microarray tell you everything? |
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Definition
| No, this doesn't test for problems with an mRNA's later actions or protein problems etc |
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