Template:SJTU-BioX-Shanghai/Notebook/Construction/w2
Construction
July 13
- Overlapping PCR to get the fragment P_dark-GFP (Annealing temperature changed to a gradience), gel analyses.</br>No correct band is showed.
- PCR for fragment PcpcB-Kn-Down in the way mentioned before to enlarge backup, gel analyses and purification.</br>Final concentration: 244ng/μl
- Inoculation of the transformed E.coli.
July 14
- Overlapping PCR for fragment P_dark-GFP. (Gradient PCR, lengthen annealing and extending time), gel analyses</br>The target band is showed but the concentration is incredibly low and extra bands existed.
- Extraction of plasmid Bluescript, gel analyses.
July 15
- PCR to get the fragment pCPCG2 from genomic DNA of cyanobacteria and Homoup-R1 from plasmid given. Gel analyses.</br>No positive results are shown. It might be the problem of template quantity or quality.
July 16
- PCR to get the fragment pCPCG2 from genomic DNA of cyanobacteria and Homoup-R1 from plasmid given or the previous fragment UP.
pCPCG2 Homoup-R1(from plasmid) Homoup-R1*(from DNA fragment) ddwater 72μl 23μl 22.5μl Template 20μl(70ng/50μl) 0.3μl(1ng/50μl) 0.5μl(<200ng/50μl) Primer1 4μl 2μl 2μl Primer2 4μl 2μl 2μl PrimeSTAR Mix 100μl 50μl 50μl Total volume 200μl(50μl*4) 100μl(50μl*2) 100μl(50μl*2) × 34 cycle 98℃ 3min 98℃ 3min 98℃ 3min 98℃ 10s 98℃ 10s 98℃ 10s (gradient) 65℃ 5s 65℃ 5s 56℃~62℃ 10s 72℃ 3s 72℃ 3s 72℃ 2s 72℃ 3min 72℃ 3min 72℃ 3min 12℃ ∞ 12℃ ∞ 12℃ ∞ Final concentration(after purification) 14.3ng/μl \ 29ng/μl *gradient PCR showed that all annealing temperature can appropriately function.
- Gel analyses for fragment pCPCG2 and Homoup-R1 and gel extraction.
July 17
- PCR for fragments Homoup2, Homoup-R1-R2 and HR(including RBS);
Homoup2 Homoup-R1-R2 HR ddwater 90μl 84μl 92μl Template DNA fragment 4μl(<200ng/50μl) DNA fragment 8μl(116ng/50μl) Synthetic plasmid 0.1μl(1ng/50μl) Primer1 4μl 4μl 4μl Primer2 4μl 4μl 4μl PrimeSTAR Mix 100μl 100μl 100μl Total volume 200μl(50μl*4) 200μl(50μl*4) 200μl(50μl*4) × 34 cycle 98℃ 3min 98℃ 3min 98℃ 3min 98℃ 10s 98℃ 10s 98℃ 10s 68℃ 5s 65℃ 5s 53℃ 5s 72℃ 3s 72℃ 4s 72℃ 5s 72℃ 3min 72℃ 3min 72℃ 3min 12℃ ∞ 12℃ ∞ 12℃ ∞ Final concentration (after purification) 83.5ng/μl 116.4ng/μl 114ng/μl
Gel analyses and purification/gel extraction.
- Overlapping PCR to get the fragments Homoup2-pCPCG2 and P_dark-HR
Homoup-pCPCG2 P_dark-HR ddwater 89μl 92μl Template1 Homoup2 0.3μl(23ng/50μl) Homoup-R1-R2 0.43μl(50ng/50μl) Template2 pCPCG2 3.5μl(50ng/50μl) HR 0.27μl(31ng/50μl) Primer1 4μl 4μl Primer2 4μl 4μl PrimeSTAR Mix 100μl 100μl Total volume 200μl(50μl*4) 200μl(50μl*4) (round 1)×8 cycle 98℃ 3min 98℃ 3min 98℃ 10s 98℃ 10s 55℃ 5s 45℃ 5s 72℃ 3s 72℃ 5s 72℃ 3min 72℃ 3min 12℃ ∞ 12℃ ∞ 12℃ ∞ 98℃ 3min 98℃ 3min 98℃ 10s 98℃ 10s (gradient) (gradient) 56℃~68℃ 5s 59℃~64℃ 5s 72℃ 3s 72℃ 8s 72℃ 3min 72℃ 3min 12℃ ∞ 12℃ ∞ Final concentration (after purification) 121.5ng/μl 187.3ng/μl
*gradient PCR showed that all annealing temperature can appropriately function.</br>Gel analyses and purification/gel extraction.
July 18
- Overlapping PCR for fragment pCPCG2-HR.
*gradient PCR showed that all annealing temperature can appropriately function.</br>Gel analyses and purification/gel extraction.PcpcG2-HR ddwater 89μl Template1 Homoup-PcpcG2 1.6μl(50ng/50μl) Template2 HR 1.4μl(41ng/50μl) Primer1 4μl Primer2 4μl PrimeSTAR Mix 100μl Total volume 200μl(50μl*4) 200μl(50μl*4) 98℃ 3min 98℃ 10s 45.5℃ 5s 72℃ 5s 72℃ 3min 12℃ ∞ 200μl(50μl*4) 98℃ 3min 98℃ 10s (gradient) 59℃~63℃ 5s 72℃ 9s 72℃ 3min 12℃ ∞ Final concentration(after purification) 145.2ng/μl
- Overlapping PCR to get the fragments P_dark-HR-PcpcB and pCPCG2-HR-PcpcB;
Gel analyses and purification/gel extraction.P_dark-HR-PcpcB PcpcG2-HR-PcpcB ddwater 80μl 80μl Template1 P_dark-HR 0.4μl(19ng/50μl) PcpcG2-HR 0.47μl(17ng/50μl) Template2 PcpcB 12μl(50ng/50μl) PcpcB 12μl(50ng/50μl) Primer1 4μl 4μl Primer2 4μl 4μl PrimeSTAR Mix 100μl 100μl Total volume 200μl(50μl*4) 200μl(50μl*4) （round 1）×8 cycle 98℃ 3min 98℃ 3min 98℃ 10s 98℃ 10s 65℃ 5s 65℃ 5s 72℃ 8s 72℃ 9s 72℃ 3min 72℃ 3min 12℃ ∞ 12℃ ∞ (round2) ×33 cycle 98℃ 3min 98℃ 3min 98℃ 10s 98℃ 10s 57℃ 5s 57℃ 5s 72℃ 12s 72℃ 13s 72℃ 3min 72℃ 3min 12℃ ∞ 12℃ ∞ Final concentration (after purification) 165.8ng/μl 170.4ng/μl
- Overlapping PCR to get the whole construction P_dark-HR* and pCPCG2-HR*
Gel analyses and purification/gel extraction.P_dark-HR* PcpcG2-HR* ddwater 88μl 88μl Template1 P_dark-HR-PcpcB 1.1μl(46ng/50μl) PcpcG2-HR-PcpcB 1μl(43ng/50μl) Template2 PcpcB-Kn 2.9μl(50ng/50μl) PcpcB-Kn 2.9μl(50ng/50μl) Primer1 4μl 4μl Primer2 4μl 4μl PrimeSTAR Mix 100μl 100μl Total volume 200μl(25μl*8) 200μl(25μl*8) （round 1）×8 cycle 98℃ 3min 98℃ 3min 98℃ 10s 98℃ 10s 68℃ 8s 68℃ 8s 72℃ 12s 72℃ 12s 72℃ 3min 72℃ 3min 12℃ ∞ 12℃ ∞ (round2) ×33 cycle 98℃ 3min 98℃ 3min 98℃ 10s 98℃ 10s 62℃ 8s 61℃ 8s 72℃ 18s 72℃ 19s 72℃ 3min 72℃ 3min 12℃ ∞ 12℃ ∞ Final concentration (after purification) 8.1ng/μl 14.9ng/μl
The final concentration is not satisfying enough because the extra band existed and gel extraction had to be operated. We adjusted several conditions and tried for a couple of days. During this time, we also changed restriction reaction site and redesigned the primers. See the final version of the protocol of these two fragments at “20150729”.
- PCR for fragments Ecolivk and sll1338 (both are signal sequences) from the synthetic plasmid. But we failed all the time. The main problems are: the concentration of the PCR products are low and for the fragments are too small (both under 120bp) to stay stably on the column when purifying, the purified products could not be used for downstream operation. We tried and adjusted constantly for at least half a month, but no satisfying results were showed.