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29 Cards in this Set
- Front
- Back
Average length of DNA fragments |
1) x the frequencies in decimals 2) = fraction (bottom no. / top no.) 3) round up that number to get average length in bp |
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Chimeric protein |
Proteins created through the joining of genes that originally coded for separate proteins |
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2-primer mutagenesis: why use restriction enzyme DpnI? |
To remove the parent template DNA |
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Why use alanine as the aa substitute during scanning mutagenesis? |
Non-bulky, chemically inert, methyl functional group that mimics the secondary structure preferences that many other aa’s possess |
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Why is non-degenerate saturation mutagenesis better than degenerate? |
Most efficient as only uses 20 codons, 1 for each of the 20 aa’s. No protein library bias, no truncation, maximises sequence space. |
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Phage display: Why does gene of interest need to be fused in frame with gene encoding the phage coat protein? |
To avoid frameshift mutations affecting the C terminal protein and resulting in a non-functional gene |
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NG pyrosequencing: How is dNTP detected? |
Specific primer annealed to template DNA and extended with DNA polymerase, 1 nucleotide is washed over template DNA at a time and if complementary, it incorporates, and 1 pyrophosphate is lost and converted in to a light signal via ATP synthesis and luciferase. |
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Techniques for working out the function of a gene of interest? |
Random mutagenesis by PCR Cassette mutagenesis Site directed mutagenesis |
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Why should specific activity of your protein increase during protein purification? |
Amount of protein (mg) is typically less but the rate of reaction stays the same (or May increase due to less interference or removal of inhibitors) |
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Stock volume calculation |
V1 = (V2xC2) / V1 1M = 1000 mM |
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Probability of a restriction site being cut by 2 enzymes calculation |
1) what bases do we need for the 2 restriction sites to be the same 2) frequency of these bases as a fraction 3) x the fractions e.g. 1/4 x 1/4 = 1/16 |
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How is ATP used in ligation? |
Ligase + ATP = Ligase-ATP + PPi AMP + phosphate = product-ligase + AMP |
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What would you substitute for glutamine? |
- charge: glutamic acid (O rather than NH2) + charge: lysine (no C=O and 2 extra CH2) Hydrophobic: methionine (similar length; 2 CH2) |
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What would you substitute for glutamine? |
- charge: glutamic acid (O rather than NH2) + charge: lysine (no C=O and 2 extra CH2) Hydrophobic: methionine (similar length; 2 CH2) |
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Megaprimer mutagenesis: what cycle is first to generate correct length product with mutation in both strands? |
Cycle 3 |
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Why is TaqMan better than SYBR green staining in QPCR? |
SYBR green binds to any, including non-specific ds DNA and so false positives may be generated. TaqMan is more specific to the gene of interest |
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Why is TaqMan better than SYBR green staining in QPCR? |
SYBR green binds to any, including non-specific ds DNA and so false positives may be generated. TaqMan is more specific to the gene of interest |
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What factors must be optimised to max production of a soluble, recombinant protein in E.coli? |
Temperature, induction point and culture time |
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What is the need for activity assays after each column during conventional chromatography? |
Ensure protein activity isn’t lost by denaturation over time or if target protein is lost during previous elation, those that lose activity can be excluded from further columns |
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2 enzymes that make both 5’ and 3’ sticky ends of DNA blunt? |
SmaI and klenow fragment |
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Methods to identify aa in active site responsible for difference specificities? |
Sequence homology - identifies the region of the protein responsible for specificity. Alanine scanning - replace each aa with alanine and examine the effect on protein and identify key aa’s. Saturation mutagenesis - introduces mutations in to a single or set of codons and creates a protein library which can be screened for proteins with required activity |
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Threonine substitutions? |
- charged: aspartic acid (H and C=O instead of CH3) + charged: lysine (NH3+ rather than OH and CH3) Hydrophobic: valine (same length, only differs by 1 CH3 which is an OH) |
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Features of DpnI |
1) recognises methylated DNA only e.g. bacterial DNA and not PCR synthesised DNA 2) 4 base recognition sequence which cuts plasmid efficiently 3) recognises small, frequent restriction sites |
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Phage display library |
Many variants of 1 protein which is screened for required activity |
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Phage display library |
Many variants of 1 protein which is screened for required activity |
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A) how many different clones theoretically in the library? B) how much seq space can library occupy? |
A) e.g. NNK = 32 codons ^7 (number of positions saturated) B) (1x10^10 / 32^7) x 100 = % |
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5 key features that prokaryotic expression vector must have (E.coli) |
Promotor, RBS, fusion domain, gene and terminator |
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Affinity tags used in affinity purification |
Maltose binding protein, glutathione S-transferase, His6 Tag |
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Anion or cation exchange? |
If pH is > pI (more basic), H+ has been removed (NH3+ -> NH2 and COOH -> COO-), protein is - and sticks to + column. pI (isoelectric point) is the pH at which the molecule has no net charge and is neutral. |