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From 2010.igem.org

Contents 1 Andreas 1.1 Site-directed mutagenesis of SOD & yCCS 1.1.1 Sequencing results 2 Mimmi 2.1 SOD / yCCS 2.1.1 Digestion

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 three 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.

Mimmi

SOD / yCCS

Digestion

pSB1C3.SOD/pSB1C3.yCCS				pMA.His			pSB1K3.RFP
8<   EcoRI + AgeI				8<   NgoMIV + PstI			8<   EcoRI + PstI
Mix	SOD	yCCS			His-tag			RFP
sH2O	7	6			14			11
DNA	19	20			12			15
10X FD buffer	3	3			3			3
EcoRI	0.5	0.5		NgoMIV	0.5		EcoRI	0.5
AgeI	0.5	0.5		PstI	0.5		PstI	0.5
tot	30µl	30µl		tot	30µl		tot	30µl

• Incubate with just NgoMIV in 37°C for 1h 30m
• Add the other restriction enzymes and incubate in 37°C for 30m
• Inactivate restriction enzymes in 80°C for 5m