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From 2010.igem.org

Contents 1 Andreas 1.1 Sequencing results 1.2 Plasmid prep 1.3 Sorting glycerol stocks

Andreas

Sequencing results

Sequencing results from 27/9 samples arrived and were analyzed by nucleotide BLAST.

Construct	Sequencing result	Blastn result	Comment
pEX.N-TAT⋅SOD⋅His 3	pEX.nTAT.SOD.his 3_premix	Blastn	Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pEX.N-TAT⋅SOD⋅His 4	pEX.nTAT.SOD.his 4_premix	Blastn	Not verified. Incorrect construct.
pSB1A2.RBS.yCCS 3	pSB1A2.RBS.yCCS 3_premix	Blastn	Partly verified. Three mutations found: (1) Single base deletion in yCCS sequence, causing a frame shift and an extended open reading frame; (2) Insertion between SpeI and PstI, not affecting expression of cloning; (3) Point mutation in PstI site, disabling PstI restriction. Mutations (1) and (3) are both quite unsure, as the sequencing quality in this area is very low. New sequencing from VR will be sent.
pSB1A2.RBS.yCCS 4	pSB1A2.RBS.yCCS 4_premix	Blastn	Partly verified. Four mutations found: (1) Point mutation changing a K codon to T; (2) Single base deletion in yCCS sequence, causing frame shift; (3) Insertion between SpeI and PstI, not affecting expression of cloning; (4) Point mutation in PstI site, disabling PstI restriction. See comment
pSB1K3.N-TAT⋅SOD⋅His 4	pSB1K3.nTAT.SOD.his4_premix	Blastn	Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pSB1K3.N-TAT⋅SOD⋅His 5	pSB1K3.nTAT.SOD.his5_premix	Blastn	Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pSB1K3.N-Tra10⋅SOD⋅His 5	pSB1K3.nTra10.SOD.his5_premix	Blastn	Partly verified. A point mutation between the NotI and XbaI sites in the prefix, not affecting cloning; mutation will be removed after XbaI digestion.
pSB1K3.N-LMWP⋅SOD⋅His 1	pSB1K3.nLMWP.SOD.his1_premix	Blastn	Partly verified. A point mutation between the NotI and XbaI sites in the prefix, not affecting cloning; mutation will be removed after XbaI digestion.
pSB1C3.N-LMWP⋅SOD⋅His 1	pSB1C3.nLMWP.SOD.his1_premix	Blastn	Not verified. Incorrect construct.
pSB1C3.N-LMWP⋅SOD⋅His 4	pSB1C3.nLMWP.SOD.his4_premix	Blastn	Not verified. Incorrect construct.
pEX.SOD	pEX.SOD_premix	Blastn	Partly verified. Insertion found between SpeI and PstI; does not affect expression.
pEX.yCCS 5	pEX.yCCS 5_premix	Blastn	Partly verified. Insertion found between SpeI and PstI; does not affect expression.
pEX.SOD⋅His	pEX.SOD.his_premix	Blastn	Verified. Three silent mutations in His tag; does not alter a.a. sequence.
pEX.His⋅SOD	pEX.his.SOD_premix	Blastn	Verified. Three silent mutations in His tag; does not alter a.a. sequence.

Plasmid prep

From 28/9 ON cultures

Spun down ON cultures 10 min, 3000 x g. Decanted supernatant and stored pellets in -20 °C ON for later plasmid prep.

Sorting glycerol stocks

Started sorting our glycerol stocks into a spreadsheet for future reference.