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Team:Stockholm/29 September 2010
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|'''<span style="color:green">Verified'''</span>. Three silent mutations in His tag; does not alter a.a. sequence. | |'''<span style="color:green">Verified'''</span>. 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. | ||
Revision as of 18:54, 30 September 2010
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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.
