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Revision as of 11:27, 20 June 2013
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Laboratory Notebook
Week 1
6/10/13
General BioMOD Cloning
- purpose: clone for DH5α pDIMC8 BlaCaM His and DH5α pDIMC8 DsbA BlaCaM
- 5 mL LB
- 100µL 20% glucose
- Cm (34 mg/mL working stock), 7.35 µL of this, final concentration 50 µg/mL
- Add cells
- Inoculate overnight
6/11/13
I. Bacterial Solution: LB Agar Plates
- purpose: to make agar plates for 1L LB-agar
- Mix stock
- 960mL DI water (860 mL if using glucose optional)
- 10g of Tryptone
- 10g of NaCl
- 5g yeast extract
- 15g agar
- Autoclave
- liquid (not dry) 30 min. sterilization/20 min drying
- cool to ~50°C in water bath
- Done under flame
- Add glucose - final concentration 20% glucose, if using 20% glucose then 100 mL, optional
- Add antibiotics to the specified final concentration. e.g. Cm: 50 μg/mL. Working stock: 34 mg/mL ⇒ 735 μL per 500 mL
- Pour onto the petri dishes, pre-mark them by type, e.g. : two black lines: Cm, red line: glucose
- Store
II. Experiment II from 6/10/13 continued: Miniprep (QIAGen)
- purpose: to purify plasmids from our clone colonies
- We followed MiniPrep
- transfer cell cultures into centrifuge tubes. Be careful not to drop the tip in.
- Centrifuge for 5 min. at 4000 rpm. N.B. make sure things are balanced.
- Dump the supernatant into old tube. Later add bleach before pouring down sink.
- Resuspend pellets and tansfer to 1.5mL labeled tube with 250μL P1 Buffer.
- 250 μL P2 Buffer (blue color): mix thoroughly.
- Time since step 2 must be less than 4 minutes: add 350μL of N3 Buffer. Mix thoroughly.
- Centrifuge at 13,000 rpm for 10 minutes.
- Apply the supernatant to QIAprep spin column.
- Centrifuge 1 minute. Discard flow-through.
- Wash w/ 750 μL Buffer PE.
- Centrifuge 1 minute. Discard flow-through.
- 1 min additional centrifuge.
- Column into 1.5 microcentrifuge tube.
- Elute with 50 μL water. 5 minute rest.
- Centrifuge 1 minute.
III. NanoDrop
- We followed NanoDrop
IV. PCR Amplification: HercII PCR
- Purpose: make backbone vectors for our Gibson reaction from pDIMC8 DsbA BlaCam. Will make:
- pDIMC8 AIDA
- pDIMC8 DsbA GLucCaM
- pDIMC8 GLucCaM His
- pDIMC8 DsbA GLucCaM His
Forward/Reverse Primer Chart:
Forward | Reverse | Key | |
---|---|---|---|
AIDA | pDIMC8 gib - for | pDIMC8 gib - rev | FW |
DsbA GLucCam | pDIMC8 gib - for | pDIMC8 DsbA gib - rev | CE |
GLucCaM His | pDIMC8 addHis - for | pDIMC8 gib - rev | TRN |
DsbA GLucCaM His | pDIMC8 addHis - for | pDIMC8 DsbA gib - rev | DZ |
- We followed Herc II PCR protocol.
Week 2
Transformed and grew up colonies of cells with CE, TN, and DZ plasmids.
- Realized that our TN plasmids were assembled with incorrect primers.
- The resulting plasmids almost certainly do not have the genes we want inserted.
- Used Qiagen MiniPrep kit to extract DNA from cells
- Sent DNA off to have our plasmids sequenced to check for correctness.
- Only send a few colonies of DZ and CE cells -- TN was not worth checking
Results:
- The DZ plasmid was correctly assembled in one of our sequences
- The other had a single mutation at residue 31 of GLucCaM gene
- One of the CE sequences was confirmed as well
- The other produced a sequence of too low quality -- likely not a correct plasmid
We started work on GLuc plasmids that contain only the G-Luciferase gene without the Calmodulin insertion.
- Assembled plasmids with the same combination of His and DsbA tags as our GLucCaM plasmids.