Team:Vilnius-Lithuania/Notebook
Notebook
09.03
Transformation of pET-Duet pET-Duet N-terminus Strep-tag C-terminus His-tag in DH5alpha
09.05
Selection of corrent colonies and inoculation in minipreps for DNA extraction.
1. Miniprep extraction • Extraction of pET-Duet pET-Duet N-terminus Strep-tag C-terminus His-tag. Concentration 30 ng/µl; • Need to cut ̴ 10 µg * with FD NcoI and FD XhoI Concentation = 7,8 ng/µl 2. psB1C3 Picked 5 – 8 pSB1C3 and ran an agarose gel: 3. Ligation with T7 polymerase
09.06
Prepare and autoclave growth medium: 20ml x2.
09.08
Minipreps purification (RFP + pSB1C3). Bacteria are pink in liquid medium. Centrifuged 10ml (in 15ml tubes). Prepared 9 RFP (1) minipreps for tomorows purification. Antibiotics Cm. We got oligonucleotides: channels (PheP), tRNR. Made PCR with a temperature gradient for PheP, tRNR biobrick, DH10B tRNR, cProm tRNR. Polymerase: Phusion.
09.09
Minipreps purification. Yesterday’s PCR succeeded. Purification with PCR purification kit. pSB1C3 + RFP restriction. RE: EamII05I, NcoI, EcoRI, PstI. Fragments purificated through column. Transformed terminal sequence (biobrick) from kit plate 3 (3F). → DH10B Insert and vector ligation. Vector: pSB1C3 Insert: PheP, tRNA B, tRNA D, tRNA C. Transform into DH10B.
09.10
Yesterday’s transformation and PheP cloning did not succeed. Colony PCR. PheP and terminator transformation. Competent cells DH5α. pSB1C3 + RFP miniprep purification. Restriction with FD EamII05I, FD NcoI, FD EcoRI, FD PstI Fragments cutting: PheP and tRNA D with FD EcoRI and FD PstI Fragments and vectors after RE purification through column Ligation. PheP into pSB1C3, tRNA with promoter DH10B into pSB1C3 PheP, tRNA D and terminator transformation. Colony PCR did not succeed. Colonies are red that means RFP remained.
09.11
Yesterday’s transformation did not succeed. Today we are repeating experiment with other RE. Vector: pSB1C3; RE: FD EamII05I, FD PstAI, FD EcoRI, FD PstI.
09.12
Miniprep DNA extraction (pSB1C3+RFP) Digest with EcoRI and PstI Colonies PCR Arrived new genes: modified gp45 is csm4. Preparation for cloning with PCR. Phusion polymerase.
09.13
Miniprep DNA extraction: YFP, RFP, PheP, tRNAB, tRNAD, tRNAC, TERM. Digestion of pSB1C3. Ligation Transformation pSB1C3+tRNAB, pSB1C3+tRNAD, pSB1C3+tRNAC, pSB1C3+PheP. PCR of GP45 and Csm4. Purification with PCR purification kit.
09.14
Preparation of minipreps. Extraction of DNA from minipreps with TERM plasmids. Ter2 digested with FD EcoRI and FD PstI.
