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: