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Team:Peking/Notebook/ALiu
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Ao Liu's Notes
Contents
June
| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
| - | 1 | 2 | 3 | 4 | 5 | 6 |
| 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | - | - | - | - |
[TOP]
6.24
Transform plasmids to Trans5a. pSB1A3, pSB1AC3, pSB1AK3, pSB1AT3, pSB1C3, pSB1K3, pSB1T3, pSB3C5, pSB3T5, pSB4C5, pSB4K5, pMAL-C4X, pET-39b.
6.26
Prepare plasmid DNA. pSB1A3, pSB1AC3, pSB1AK3, pSB1AT3, pSB1C3, pSB1K3, pSB1T3, pSB3C5, pSB3T5, pSB4C5, pSB4K5, pMAL-C4X, pET-39b
Digest the plasmid DNA with EcoRI &PstI.
Retrieve the digested product
6.27
Transform plasmids to Trans5a.1-12O, pSD-MBD, pASK-MBD, NRI.
6.28
Picking colonies and shaking at 37℃ overnight. 1-12O
Prepare plasmid DNA. pSD-MBD, pASK-MBD, NRI.
6.29
PCR Strain NRI with two pairs of primers
6.30
PCR Strain NRI with two pairs of primers
Retrieve the PCR product
Prepare plasmid DNA. 1-12O
July
| Mon | Tue | Wed | Thu | Fri | Sat | Sun |
| - | - | - | - | 1 | 2 | 3 |
| 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 11 | 12 | 13 | 14 | 15 | 16 | 17 |
| 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| 25 | 26 | 27 | 28 | 29 | 30 | 31 |
[TOP]
7.1
Digest the plasmid DNA with EcoRI &XbaI. 1-2M, MerR, E0840
Retrieve the digested product
7.17
Transform plasmids to Trans5a. merP(mutant)-E0840, 1-18I-MerR, merP-E0840.
7.18
Transform s merP-E0840 and 1,18I-MerR to Mach-1.
7.19
PCR to build merT promoter mutants library
Retrieve the PCR product
Digest the PCR product by EcoRI and PstI
Induce merTP-E0840+1-18I-MerR by 1E-5M Hg(II)
Retrieve the digested product
7.20
Connect digest product with pSB3K3.
7.21
Pfu-library Picking colonies and shaking at 37℃ overnight.
Taq-library Failed. Connect digest product again.
Digest the PCR product by EcoRI and PstI
Retrieve the digested product
Digest the PCR product by EcoRI and PstI overnight.
7.22
Prepare plasmid DNA. The library.
Retrieve the digested product. Rbs-MerR. Failed and digest again.
Retrieve the digested product
Connect the digest product overnight.
7.23
Digest the PCR product by EcoRI and PstI. Rbs-MerR
Connect digest product with I-18C overnight.
7.24
Retrieve the digested product
Connect digest product with I-18C overnight.
Picking colonies and shaking at 37℃ overnight.1-18C-rbs-MerR
Transform the connect product to Mach-1.
7.25
Prepare plasmid DNA. Failed
PCR to build merT promoter mutants library
7.26
Transform the plasmid of library to E.coli containing 1-18I-MerR.
7.27
Picking colonies and shaking at 37℃ overnight. The library.
Connect MerR with rbs
Transform the connect product to Mach-1.
7.28
Picking colonies and shaking at 37℃ overnight. rbs-MerR.
Activate every mutant in the promoter library until the OD600 was between 0.4 and 0.6.
Then induced them by 10^-5M Hg(II), 2 hours. Centrifuged and resuspensed with PBS.
GFP intensity and OD600 were measured by Tecan Microplate Reader.
7.29
Prepare plasmid DNA. Rbs-MerR
Activate every mutant in the promoter library until the OD600 was between 0.4 and 0.6.
Then induced them by 10^-6M Hg(II), 2 hours. Centrifuged and resuspensed with PBS.
GFP intensity and OD600 were measured by Tecan Microplate Reader.
7.30
Sequenced, rbs-MerR.
Activate every mutant in the promoter library until the OD600 was between 0.4 and 0.6.
Then induced them by 10^-6M Hg(II), 2 hours. Centrifuged and resuspensed with PBS.
GFP intensity and OD600 were measured by Tecan Microplate Reader.
7.31
Activate every mutant in the promoter library until the OD600 was between 0.4 and 0.6.
Then induced them by 10^-7M Hg(II), 2 hours. Centrifuged and resuspensed with PBS.
GFP intensity and OD600 were measured by Tecan Microplate Reader.
