Lab Notes
[Abbreviation notes: 4L = part BBa_K325909 (LuxCDABEG), 2D = part BBa_K325219 (Red firefly luciferase and LRE), 4N = part BBa_K325100 (EPIC firefly luciferase and LRE); Concentration of plasmids are in units of ng/μL (if not mentioned)]
July
7/17
- I7 test tubes, 4 monoclone (4L, 2DH, 2DH-5)
- 2D and 4N transformation (2DH,2DH-5)
7/18
- Plasmid extraction for 7 test tubes
- PCR 4L and LuxG (failed)
7/19
- Plasmid extraction
- Spreading bacteria onto petri dish:
7/21
- Four Agrobacteria transformations for 2*4L, 1*2D and 1*4N
7/25
- Transformed 2*LuxG, 2*4L, 2*2D, 2*4N, totaling up to 9 petri dishes
- 2*LuxG 2*4L 1*2D 1*4N totaling up to 6 test tubes
- Preparing petri dish for transformation:
7/26
- PCR LuxG with primers
- Added 4L monoclone into 2 Erlenmeyer flasks
- Used XBaI and PstI to digest the 4L monoclone
- Extracted plasmids (1*4N, 1*2D, 2*4L, 2*LuxG)
- Gel electrophoresis the products from PCR and enzyme digestion.
7/27
- LuxG gel extraction (digested by XBaI and PstI)
- Tested for concentration
7/28
- Took 20ml of 4L from the Erlenmeyer flask
- Extracted plasmid (4*2D, 3*4L)
- Conducted gel electrophoresis to confirm plasmid size
- Tested for concentration
- 4L succeeded, but the concentration of 2D was too low for gel electrophoresis
- Added 8mL of 0.1M Arabinose into a 80mL flask and 10ml of 0.1 Arabinose into a100mL flask
- After 3.5 hours, visible light was emitted
- Gradient test of 4L with 0.1M Arabinose
- The calculation for the preparation of different concentrations of arabinose:
- The glowing bacteria observed in cylindrical flasks:
7/29
- Prepared 3L of LB, but had to be redone due to an inaccurate electronic balance
- Prepared 2L liquid LB, 1 L solid LB. All sterilized.
- Washed and sterilized 62 test tubes.
- Organized plasmids and DNA
- Designed further experiments with guidance professors.
August
8/1
- Extracted 8 tubes of pHB plasmid
- Concentration: 67.439, 72.8, unknown, 128.6, 203.2, 153.6, 101.2, 61.7
- Digested pHB using BamHI and PstI, ran gel electrophoresis and did gel extraction
- Digested 4L using BamHI and PstI, ran gel electrophoresis and did gel extraction
- Monoclone 4L into 2 flasks and 4 test tubes
- Gradient test
- 0.1M solution emitted the brightest light.
- Gel extraction process:
8/2
- Extracted 6 test tubes of 4L plasmid
- Ligation of pHB with LuxCDABEG
- Transformation of the ligated pHB-LuxCDABEG on 2 petri dishes
- Conducted gradient test for 0.1 and 0.2M Arabinose
8/4
- Prepared 39 test tubes of 4L for gradient test the next day
- Extracted pHB-LuxCDABEG and verified it with gel electrophoresis
- Prepared 8 test tubes of pHB-LuxCDABEG monoclones
- Gel electrophoresis of pHB-LuxCDABEG:
8/5
- Extracted 8 tubes of pHB-LuxCDABEG
- Verified with gel electrophoresis, 2 bands at 7kb and 11kb are present.
- Suspected that the ligation failed
- Digested pHB with BamHI and PstI, gel electrophoresis to check whether digestion was successful (it was)
- Prepared 50mL of 2M and 1.5M Arabinose, 200mL of 1M Arabinose
- Prepared 500mL of solid YEB
- Gradient test of 4L with Arabinose at concentrations 0, 0.01, 0.1, 0.15, 0.2M
- Add Arabinose (final concentration 0.1M) into two 2L flasks with 4L for observation
- The glowing bacteria observed in cylindrical flasks:
8/6
- Gel extraction of pHB (5 tubes)
- Concentrated 5 tubes of pHB and 3 tubes of LuxCDABEG (digested by BamHI and PstI)
- Ligated pHB and LuxCDABEG
- Transformed pHB-LuxCDABEG on 2 petri dishes
8/8
- PCR LuxCDABEG with primers
- Prepared 600mL LB and separated it into 80 test tubes
- Prepared 8 tubes of monoclone pHB
- Extracted ligated pHB-LuxCDABEG
- Setting the PCR machine:
8/9
- All 80 test tubes of LB and 8 tubes of monoclone pHB are contaminated
- Prepared 1L LB and separated it into 80 test tubes; Stored the rest in a 500mL jar
- PCR LuxCDABEG with primers
- Digested 4L with BamHI and PstI, gel electrophoresis
- Prepared 8 tubes of monoclone pHB
- Concentrated yesterday’s LuxCDABEG (32, 66 ng/μL)
8/13
- Gel electrophoresis twice (pHB-LuxCDABEG) but both trials failed.
8/16
- Colony PCR (900bp, 29 tubes)
- Digested pHB and 4L (BamHI and PstI), 6 test tubes each
- Gel electrophoresis and PCR of 4L
- Gel extraction of 4L
- Washed 80 test tubes
- Prepared 400mL LB into 80 test tubes, and sterilized them.
- Put monoclones into 3 tubes of 4L, 3 tubes of pHB, 6 tubes of pHB-LuxCDABEG
8/17
- Concentrated 2 tubes 4L (concentration 3.6 and 5); combined two tubes into 1.
- The final concentration is 10
- Prepared 500mL liquid YEB and 250mL solid YEB; Sterilized them.
- Extracted plasmid (3 tubes 4L, 3 tubes pHB, 6 tubes pHB-LuxCDABEG)
- Gel electrophoresis of pHB-LuxCDABEG
- Result seems like 19kb, tomorrow need to conduct PCR and sequencing
8/18
- PCR 1 test tube of pHB and 5 test tubes of pHB-Lux
- Conducted successful electrophoresis
- Digested LuxG and pHB-Lux with HindIII and BamHI, 4 test tubes each.
- Gel Extract LuxG and pHB-Lux, 1 test tube each;
- Concentration of LuxG: 132; Concentration of pHB-Lux: 101.
- Put 5ul LuxG and 12ul pHB-Lux under 16°C overnight Ligation.
- Transform pHB-Lux into Agrobacterium (2×50ml conical flask)
- Gel electrophoresis of pHB:
8/19
- PCR 4 tubes of pHB+LuxG primer
- Conducted successful Gel electrophoresis.
- Transformed pHB-Lux-LuxG into E. coli (4 petri dishes)
- Digested pHB-Lux with BamHI and PstI
- Gel electrophoresis (6.4kb and 11kb)
8/20
- Put 34 monoclones on one petri dish
- Transformed pHB-LuxCDABEG on 4 petri dishes
- Monocloned pHB-Lux and pHB, 6 test tubes each.
8/21
- PCR 8 tubes of LuxG with primers; Gel electrophoresis failed
- Detected Agrobacteria infection on two tobacco plants
- Prepared 250mL of solid YEB, 500mL of liquid YEB, and 50mL of liquid YEB in 5 flasks each; Sterilize all.
- Add Monoclone into 50ml YEB conical flask
- Extract 5 tubes of pHB-Lux, 5 tubes of pHB, totaling up to 10 test tubes.
- 2 test tubes are contaminated and abandoned.
- Injection of agrobacteria into tobacco plants:
8/24
- Gel extraction of 2 test tubes of LuxG, with concentrations 63 and 75.
- Digested 1 tube of LuxG (BamHI, HindIII), gel electrophoresis and gel extraction
- Ligated LuxG (BamHI, HindIII) and pHB-Lux (BamHI, HindIII)
8/27
- Watered plants
- PCR of pHB-Lux-LuxG
- Got monoclone of 4L Cl+
- Ran electrophoresis for the PCRed DNA
- Got monoclone for pHB-Lux-LuxG
- Prepared 500 mL LB
8/28
- Extraction of plasmids of 4L and pHB-Lux-LuxG
- Results of Concentration test: 4L:300-400, pHB-Lux-LuxG: 20-40
8/29
- Electrophoresis of pHB-Lux-LuxG received no results.
- Watered Plants
- No glowing is observed in plants.
8/30
- Transformed 4L
- Digested pHB-Lux and LuxG
- Ligated overnight
- Checked on plants
September
9/7
- Washed 70 test tubes
- Prepared 1L LB into 70 test tubes and 3*250mL jars; Sterilized
- Digested pHB-Lux using BamHI and PstI, gel electrophoresis
- Monoclone 4L into 250ml flask
- Gel electrophoresis of pHB-Lux:
9/8
- Prepared 1L LB, 4 250 mL bottle with 4g Agar each
- PCR LuxG with primer and checked with electrophoresis but received no results.
- Added 20μL of 4L and 5μL Cl+ antibiotic each into 70 test tubes.
- Digested pHB-Lux (BamHI, HindIII) into 4 tubes
- pHB-Lux in 2 test tubes are extracted directly, and 2 others went through gel extraction.
9/9
- LuxG PCR with SpeI and PstI primers (8*50 μL)
- Gel extraction for:
LuxG (after electrophoresis), 3*50 μL
4L-SpeI/PstI (1*50 uL);
LuxG-SpeI/PstI (1*50 uL)
- Restriction enzyme digestion (using SpeI and PstI) for LuxG and 4L (both 4*25 μL)
- Overnight ligation of LuxG and 4L
- Gradient test of 0M, 0.01M, 0.1M, 0.15M, 0.2M
- Time: 0-5hrs, 1 measurement per hour
- Preparing the 96-well plate for gradient test:
9/15
- LuxG pHB-Lux ligation 2*20μL for 4 hours under 25°C (BamHI, HindIII)
- Transformation of pHB-lux-LuxG (2 plates LB, Kan+)
- Transformation of pHB-LuxG (3 plates,YEB, Kan+ Rif+) 4L LuxG ligation 3*20μL (overnight 16°C)
- Pick single colony unit 4L-LuxG (8 tubes, LB, Cl+)
9/21
- Colony PCR for pHB-Lux-LuxG and pSB1C3-Lux-LuxG
- Electrophoresis of Lux-LuxG plasmid to verify whether ligation was successful
- Gel electrophoresis of 4L-LuxG and 4L-LuxG-pHB
- Preparation of electrophoresis:
