| Column 1 | Column 2 (memorize)
|
| Gateway cloning technology | 1). Insert the gene of interest into an entry clone (gateway), where the gene of interest is flanked by attL recombination sites. 2). In the LR reaction, the sticky ends of from the digestion of the attL sites in the entry clone will match the sticky ends produced by the digestion of the attR restriction sites of the expression clone. 3) It can be transfered using combination of enzymes and transforming E. coli.
|
| Lambda recombination system | A basic variation on gateway cloning technology.
|
| Site-directed mutagenesis | Design a primer that you want to introduce. After amplification of the DNA with this primer, the DNA fragment should have this specific mutation. Verify with sequencing.
|
| Northern blotting | It is a technique used to study gene expression by detection of RNA (or isolated mRNA) in a sample. A unique advantage is that it can detect transcript size. However, Northern blot is not very sensitive and requires a relatively large amount of mRNA.
|
| Real-time PCR | It is the most sensitive method of detecting mRNA or DNA. In order to detect mRNA, it needs to be converted to DNA using RT-PCR. The cost is low to moderate ($200-$500).
|
| DNA microarrays | DNA sequences (probes) are covalently linked on the chemical matrix of a solid surface by surface engineering and targets hybridize to these probes under stringent conditions. Well suited to exploratory studies, since it is fast but the results can be difficult to interpret.
|
| SAGE / High-throughput cDNA sequencing | Serial analysis of gene expression - on its way out. High-throughput cDNA sequencing - may become gold standard. The minimum uniquely-identifying sequence is extracted for each gene. Cost is high. Sensitivity is high. Gives absolute levels of each transcript.
|
| Western blotting | Tests protein expression. Separate proteins by denaturing gel electrophoresis. Incubate with primary antibody. Incubate with HRP-coupled secondary antibody. Detect with chemiluminescent substrate. Wash and expose to X-ray film. Fast, low-cost. Many antibodies that work for other applications don't work for Western and vice versa.
|
| Antibody arrays | Print array; hybridize with labeled protein sample; wash; and scan. Fast, moderate-high cost. Can detect many proteins without expensive equipment. Technology is currently crude.
|
| Mass spectrometry | Separation of proteins on a 1-D or 2-D gel, excision of the gel fragment that contains the protein of interest, extraction of the protein, fragmentation, ionization and separation by mass spectrometry. Can detect posttranslational modifications.
|
| What is the basic DNA manipulation technique for cutting? | Restriction enzymes.
|
| What is the basic DNA manipulation technique for copying? | Reverse transcriptase.
|
| What is the basic DNA manipulation technique for pasting? | DNA ligase.
|
| What is the basic DNA manipulation technique for detecting? | Southern blotting.
|
| What is the basic DNA manipulation technique for amplifying? | PCR.
|
| What is the basic DNA manipulation technique for reading? | DNA sequencing.
|
| What is the basic DNA manipulation technique for rewriting? | Site-directed mutagenesis/gene synthesis.
|
| What is the best technique to deliver a single copy of a construct of interest to neurons in culture? | Viral transduction.
|
| What is the best way to comprehensively annotate and quantify all mRNAs expressed in the PC12 cell line? | SAGE
|
What is the best way to directly visualize lysosome dynamics in living tissue? ||
