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.
09.15
Prepared minipreps (pET-Duet, pET21b). Extract TERM2 from minipreps with DNA extraction kit. Digest pSB1C3. FD EcoRI with FD PstI Purification of pSB1C3. Digestion of pETDuet and pET21b with XhoI and NcoI Ligation and transformation of PolyPhe proteins.
09.16
pSB1C3-TERM-PheP, tRNAC and D colonies PCR. Cloning modified Gp45 into pET. Transformation of PheP, tRNA into pSB1C3.
09.17
Colonies PCR: Csm4 in pET and Csm4, Gp45 in pSB1C3 Extracted plasmids: pSB1C3-TER-PheP, pSB1C3-TER-tRNAD, pSB1C3-TER-tRNAC. Colonies PCR: GP45 in pET.
09.18
Extraction DNA from minipreps
09.19
Cloning Gp45 into pSB1C3, Csm4 into pET. Prepared minipreps of pSB1C3-PheP-tRNAB, pSB1C3-PheP-tRNAC, pSB1C3-PheP-tRNAD PCR of plasmids.
09.21
Transformation pSB1C3-Gp into DH5alpha.
09.24
Check Gp in pSB1C3 with colonies PCR. Digest Csm for cloning into pET. Sequencing results of Gp, PheP, tRNAC, tRNAD.
09.25
DNA extraction pET with Csm prepared for cloning. Ligation with T7 polymerase.
09.26
Transformation of PheP with tRNA. Cloning of Csm into pET. Prepared samples for sequencing.
09.27
Extraction of DNA from minipreps Csm in pET. Cloning of PheP into pSB1C3+term+tRNA. Digestion of Csm in pET, tRNA+PheP.
09.28
SDS-PAGE+NB of GP45 modified genes.