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Integration Primer Set |
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Prices are in US dollars and valid only for US orders.
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Description:
Integration Primers 1, 2 and 3 are components of the K. lactis Protein Expression Kit (NEB #E1000). They are used in whole-cell PCR reactions to detect integration of single and multiple tandem copies of the expression vector pKLAC1 into the K. lactis chromosome (Figure 1).
Integration Primers 1 and 2 will amplify a 1.9 kb DNA fragment if integration of pKLAC1 has occurred correctly at the LAC4 promoter locus on the chromosome (Figure 1, part A).
It is possible for multiple copies (up to ~ 10) of pKLAC1 to insert in tandem into the genome during transformation of K. lactis GG799 Competent Cells (NEB #C1001) followed by acetamide selection. Multiple integrants can be identified using whole-cell PCR with Integration Primers 2 and 3 to amplify a 2.3 kb DNA fragment (Figure 1, part B).
Figure 1: Integration of linearized pKLAC1.Upon transformation of K. lactis cells with the linearized expression vector, the fragment inserts into the promoter of the LAC4 locus. Single-copy integration can be detected by PCR using Integration Primers 1 and 2 to amplify a 1.9 kb diagnostic fragment (A), whereas multi-copy integration can be detected using Integration Primers 2 and 3 to amplify a 2.3 kb fragment (B).
Reagents Supplied:
Integration Primer 1 (10X)
Integration Primer 2 (10X)
Integration Primer 3 (10X)
Storage Conditions
Storage Temperature:
-20°C
Notes
General notes:- Integration Primer Set contains 500 μl of Integration Primer 1, 1,000 μl of Integration Primer 2 and 500 μl of Integration Primer 3.
Companion Products
K. lactis GG799 Competent Cells
K. lactis Protein Expression Kit
BstXI
pKLAC1 Vector
pKLAC1-malE Control Plasmid
SacII
Yeast Medium Pack
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