Slide3 - www.stratagene.com Map of Bluescript plasmid...

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1/10/10 1 As of 2008, 100 gigabases of sequence information in GenBank (100 billion bases) http://www.ncbi.nlm.nih.gov/ Genbank/genbankstats.html Tm is proportional to G+C Content Relative absorbance (260 nm) Temperature (C) 70 80 90 100 1.4 1.2 1.0 Pneumococcus (38%) G+C E. coli (52%) S. marcescens (58%) M. phlei (66%) Adapted from Garrett & Grisham (1999) Biochemistry (2e) p.372 Tm becomes higher as ionic strength of solution is increased Adapted from Garrett & Grisham (1999) Biochemistry (2e) p.372 60 70 80 90 100 110 100 80 60 40 20 0 Tm (C) G + C (%) 0.15 M NaCl 0.015 M Na citrate 0.01 M phosphate 0.001 M EDTA 3’ 5’ 5’ 3’ Index of restriction enzyme recognition sequences New England Biolabs www.neb.com
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1/10/10 2 Map courtesy of Stratagene
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Unformatted text preview: www.stratagene.com Map of Bluescript plasmid www.stratagene.com The multi-cloning site (MCS) of pBluescript www.neb.com Portion of a bacterial expression vector 1. Gene of interest cloned into MCS, in frame with bacterial malE gene 2. Bacteria make fusion protein (malE fused to your protein of interest) www.neb.com In vitro purification of fusion protein 1. Gene of interest cloned into MCS, in frame with bacterial malE gene (malE encodes maltose binding protein (MBP)) 2. Bacteria make fusion protein (MBP fused to your protein of interest) 3. Purify fusion protein using bacterial portion as a tag. In vitro purification scheme 1/10/10 3 The multi-cloning site (MCS) of pBluescript www.stratagene.com...
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Slide3 - www.stratagene.com Map of Bluescript plasmid...

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