Team:Stockholm/19 August 2010

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Revision as of 10:17, 19 August 2010

Andreas

Site-directed mutagenesis of SOD & yCCS

Sequencing results

Received the sequencing results for m-SOD 1, m-SOD 2, m-yCCS 1 and m-yCCS 4 from Eurofins. Ran a nucleotide BLAST (BLASTn) alignment to verify successful site-directed mutagenesis.

SOD 1: Failed (BLASTn results)
- Mutation still present
SOD 2: Success (BLASTn results)
- Mutation removed
yCCS 1: Success (BLASTn results)
- EcoRI site removed
yCCS 4: Success (BLASTn results)
- EcoRI site removed

All sequencings revealed an insertion in the Freiburg suffix, between SpeI and PstI restriction sites. We were already aware of this, and this will be taken care of later by digestion and cloning with SpeI, thereby removing the insertion.
BLASTn results also revealed 4 mutations in both yCCS sequences. A protein BLAST (BLASTp) alignment of the translated yCCS sequences revealed all four mutations as silent mutations (BLASTp results).

SOD 1 sample and glycerol stock will be discarded; the other clones are saved as verified.

@@ Line 15: / Line 15: @@
 *'''yCCS 4:''' Success ([[media:BLASTn_m-yCCS4_19aug.txt‎|BLASTn results]])
 **EcoRI site removed
+All sequencings revealed an insertion in the Freiburg suffix, between SpeI and PstI restriction sites. We were already aware of this, and this will be taken care of later by digestion and cloning with SpeI, thereby removing the insertion.<br />
+BLASTn results also revealed 4 mutations in both yCCS sequences. A protein BLAST (BLASTp) alignment of the translated yCCS sequences revealed all four mutations as '''silent mutations''' ([[media:BLASTp_m-yCCS1--yCCS_19aug.txt‎|BLASTp results]]).
-SOD 2 sample and glycerol stock will be saved, while SOD 1 will be discarded.<br />
+SOD 1 sample and glycerol stock will be discarded; the other clones are saved as verified.
-Unfortunately, BLAST results also discovered 4 (!) identical mutations in both yCCS sequences. A protein BLAST (BLASTp) alignment of the translated yCCS sequences revealed all four mutations as '''silent mutations'''.