2009/7/24 10:30 There are six clones on the plate. Shake those clones in the incubator. PCR clones to test if they are correct.
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1 2009/7/23 11:30 Recycle all the inserts of SupD and terminator. 12:20 Digest high-copy bi-stable plasmids. 8μL 8μL 12:30 Start to digest. 12:40 Ligation of SupD+terminator and 1-18A (strongest promoter). Vectors Inserts 7μL T4 ligase 16:50 Electrophoresis to recycle the high-copy bi-stable digestion products. The order and the amount of the samples: control plasmids, digestion products, marker 5μL. 2009/7/24 10:30 There are six clones on the plate. Shake those clones in the incubator. PCR clones to test if they are correct. Template Clones
2 dntp Taq For-primer Rev-primer 5.5μL The results are not very good. 14:00 Transform the plasmids PKD46 from France in two tubes. 22:40 Miniprep 1-18A + SupD + terminator. Number of the plasmids Concentration(ng/μL) Colony Colony Colony Colony Colony Colony /7/28 00:20 Digest 1-18A + SupD +terminator plasmids to test if they are correct. Digest them into vectors as well. Digest into inserts: 14μL Digest into vectors: 3μL 13μL PCR at the same time: Template For Rev
3 Mix 5μL 3.5μL 1:00 Start to digest. 1:30 Start to PCR. 12:00 Electrophoresis to test if the PCR products and digestion products are correct. The order and the of the samples: marker, PCR products 1, PCR 2, PCR 3, PCR4, PCR5, PCR6, digestion control plasmids, digestion1, digestion2, digestion3, digestion4, digestion5, digestion 6. 16:00 Transfrom pkd3, pcp 20 to DH5a. Digest 1-18C into vectors. 3μL 13μL 20:00 Electrophoresis the digestion products: The order and the amount of the samples: marker, plasmids control, digestion 1 15μL, digestion2 15μL.
4 The digestion products are correct. 2009/7/29 10:00 Recycle the digestion products. Miniprep the plasmids of 1-18C and PYFP. 13:30 Digest plasmids PYFP from France. Kpn1 4μL 1 13:48 Start to digest. 15:00 Transform psp20 into DH5a. Ligation of 1-18C backbone and SupD+ terminator inserts. 15:30 PCR to standardize the PYFP plasmids. Take a gradient from 54 centigrade to 58 centigrade. 50μL Template 0. dntp 4μL Phusion For-primer 1.25μL Rev-primer 1.25μL 32.8μL 17:30 Digest the high-copy bi-stable PCR products. Fragments 5μL Kpn1 19:30 Electrophoresis the digestion products and PCR products. The order of the samples: marker, plasmids control, digestion products, PCR products under 54 centigrade, PCR products under 56 centigrade, PCR products under 58 centigrade.
5 Digestion products are weird, while the PCR products under 58 centigrade are specialized. As a result, recycle the PCR products under 58 centigrade. 21:55 Digest PYFP plasmids. 5μL Kpn1 2009/7/30 9:00 Electrophoresis to test the products of the digestion. The order of the samples: marker, plasmids control, digestion products.
6 It is difficult to tell the digestion fragments, maybe because of the poor concentration of the plasmids. 20:00 Digest the PYFP plasmids into vectors. Digest to standardize: 7μL 9μL Digest to connect with high-copy bi-stable fragments. Kpn1 6μL Digest the standardizing PCR fragments into inserts: 17μL Xba1
7 Digest T7P M to get the high-copy backbone. 4μL Xba /7/31 10:30 Electrophoresis the digestion products. The order of the samples: marker, digestion products for backbone, single digestion products of the standardized fragments, double digestion products of PYFP cut by Kpn1 and, double digestion products of PYFP cut by and. Recycle the fragments. 12:00 Digest the single digestion products of PYFP by another enzyme. 30μL Fragments 25.5μL Apal1 1.5μL 3μL 12:40 Start to digest. 14:00 Miniprep. Number of the plasmids Concentration(ng/μL) PKD High-copy bi-stable PKD Psp
8 PYFP :00 Digest high-copy bi-stable plasmids. Xba1 Nhe1 15μL 16:30 Start to digest. 16:30 Start to recycle the fragments digested by ApaL1 17:30 Link the standard fragments with PYFP vectors, also high-copy bi-stable fragments with PYFP vectors. Vectors Inserts 6μL T4 ligase 22:00 Electrophoresis to test the digestion products. The order of the samples: marker, high-copy control plasmids, digestion products. Digestion is successful. Recycle the fragments of about 3kb. 22:30 Transform the ligation products into DH5a. 2009/8/1 00:30 Start to incubate. 13:30 There is no colony on the plates. 21:00 Digest the high-copy bi-stable plasmids and low-copy bi-table plasmids. High-copy bi-stable plasmids: 13μL Low-copy bi-stable plasmids: 7μL Nhe1
9 PYFP: Kpn1 8μL 6μL 6μL Digest T7P M for backbone: 4μL 1 22:00 Start to digest. 2009/8/2 1:00 Electrophoresis to test the digestion products. The order of the samples: marker, high-copy plasmids, high-copy digestion products, low-copy plasmids, low-copy digestion products, PYFP plasmids, PYFP digestion products by EK, PYFP digestion products by SP, T7P+1-12M plasmids, T7P+1-12M digestion products.
10 Electrophoresis the fragments used in the ligation the day before. The concentrations of the fragments are too low, so that cause the ligation to fail.
11 2:00 Recycle the fragments. 4:00 Connect the bi-stable fragments, standard fragments with PYFP vectors. Connect the high-copy bi-stable fragments and low-copy bi-stable fragments with the high-copy backbone. Vectors Inserts 6μL T4 ligase 13:00 Transform those ligation products into DH5a. 2009/8/3 8:30 There are many colonies on all the plates. 9:30 Start to shake the colonies in the incubator. 11:30 PCR to test the colonies at the same time. Template Colonies For Rev Mix 8μL 17:00 Electrophoresis to test the PCR products. The order of the samples: standardized PYFP, marker, PYFP with bi-stable parts, high-copy bi-stable parts, marker, low-copy bi-stable parts. 23:00 Miniprep. Number of the plasmids Concentration(ng/μL) PYFP with bi-stable parts PYFP with bi-stable parts PYFP with bi-stable parts PYFP with bi-stable parts Low-copy bi-stable parts Low-copy bi-stable parts High-copy bi-stable parts High-copy bi-stable parts PYFP with standardized fragments /8/4 2:30 Digest and PCR those plasmids to test if they are correct. PYFP with bi-stable parts: 3μL
12 Kpn1 13μL High-copy and low-copy bi-stable parts with high-copy backbone: 3μL 13μL PYFP vectors with standardized fragments: 6μL This is to test if the restriction enzyme sites are successfully been erased. PCR to test standardization: Template For Rev Mix 7μL 10:30 Electrophoresis to test if the PCR and digestion products are correct. The order of the samples: Marker, PYFP with bi-stable parts plasmids control, PYFP with bi-stable digestion products 1,2,3,5, PCR products of standardization, the digestion products of standardization. Marker, low-copy bi-stable parts, high-copy bi-stable parts.
13 The PFYP with the bi-stable parts are correct. 16:00 Make up SOB. 100ML 2009/8/5 10:00 Prepare competent cells for electrical transformation. 20:00 Digest the PYFP with bi-stable parts to get the inserts. 50μL Sap1 1.5μL Sph1 1.5μL 5μL /8/6 12:30 Recycle the digestion products. 13:30 Prepare SOC. 20:00 Electroporation: Parameters: 50μLwith 400ng DNA fragments. 2.5kV, 25mF, 200ohms. Incubate in SOC for 2h. 2009/8/7 There is one colony on the plate, but it is the result of reconnection of the plasmids. 2009/8/16 Test if the tetr promoter system works well in the low-copy backbone.
14 Get tetr+promoter+grp from Wu Shuke. 16:40 Digest the promoter and reporter system. 50μL 5μL 1 20:00 Electrophoresis to recycle the inserts. The order of the samples: marker, digestion products, plasmids control. Only the first one is correctly digested, recycle them. 2009/8/17 10:00 Link the inserts with vectors with has Kanamycin resistance. 17:50 Transformation. 19:30 Start to incubate. 2009/8/18 10:00 There are many colonies on the plate. PCR colonies to test if they are correct. 20:00 Pick the colonies to shake in the incubator. 2009/8/19
15 9:30 Mniprep the plasmids. The general concentration of the plasmids are about 150ng/μL. 11:00 Digest and PCR those plasmids to test if they are correct. The digestion system: 6μL The PCR system: Template Colonies For Rev Mix 5μL 3μL 12:00 Start to digest&pcr. 16:30 Electrophoresis to test the digestion and PCR products. All the clones have correct colonies. 17:00 Induce the strain containing tetr and low-copy backbone by atc.
16 22:00 Using flow cytometry to test the induction results. There are about 5 folds between the induced sample and the uninduced one. 2009/8/20 Make a gradient to test the features of the tetr promoter system. The gradient is from 10 2 to 10 6, control, negative control 10 2, :00 Recycle LacP vectors. Miniprep Sal promoter + SupD + terminator. 18:40 Link the LacP vectors to T7ptag inserts. Vectors Inserts T4 ligase 19:00 Ligation starts. 20:30 Transformation 23:00 Start to incubate. 7μL
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