Team:ECNUAS/Notebook
Notebook
2021-7-15
- Ice-breaking
- Initial Discussion on the project plan
2021-7-16
- Subject background survey
- Basic experimental operation
- Laboratory safety training
- Prepare growing medium (liquid culture medium)
2021-7-17
- Basic operate training
- Learning the concept of recombinant plasmids
- Prepare growing medium (solid culture medium)
• One type of Kanamycin
• One type of Ampicillin
• One type of Kanamycin and Ampicillin
- Dilute TAE buffer
2021-7-18
- Restriction endonuclease reaction
- DNA agarose gel electrophoresis identification
- DNA agarose gel reavers and purification
- DNA clip connection reaction
- Plasmid transformation
• Plasmid A (Atz sensor plasmid):pET28a(+)_P7_AtzR
• Plasmid B (Atz reporter plasmid): pUC57-pro_Pprovoin5_amilGFP
2021-7-19
- Found the plasmid B’s plate didn’t grow any colony, but Plasmid A grew
- Amplification of Atz sensor plasmid and leave it in shaker through night
- Then we start to think why plasmid B didn’t grow any flora
• We think that the plasmid transformation in day 7-18 might got some problem, so we do the DNA clip
connection and Plasmid transformation again by using day 7-18 DNA clip connection reaction.
- So we repeat experience again (but we didn’t get good stripes)
• restriction endonuclease reaction (only Pprovoin5)
- Increase the amount of sample to 15 μL
- Increase the amount of Pprovoin5 enzyme to 1.25 μL
- Increase the amount of Pprovoin5 enzyme to 1.25 μL
• DNA agarose gel electrophoresis identification (all result get very light stripes)
1. First gel is from day 7-18 DNA clip connection reaction
2. Second gel is from day 7-19 restriction endonuclease digestion product
- The potential cause why we don’t get stripes or very light stripes
A. turnip the gel 180 (may cause the stripe light)
B. When we’re adding more TAE, the TAE added might disperse light
C. Adding too much sample might disperse the stripes (because we change from 10 μL to 15 μL
B. When we’re adding more TAE, the TAE added might disperse light
C. Adding too much sample might disperse the stripes (because we change from 10 μL to 15 μL
- Preparing LB medium
2021-7-20
- Continue plasmid extraction
• By using the colonies from
day 7-19 (plasmid A)
- Restriction endonuclease reaction and DNA agarose gel electrophoresis
identification (only Pprovoin5) (twice because first one has no stripe)
1. No change
2. Changing the system from
50μL to 40μL by decrease water (10μL)
- DNA agarose gel electrophoresis identification result
1. No/very light stripe
2. Stripe appeared
2. Stripe appeared
- DNA clip connection reaction
- Plasmid transformation
A. Restriction endonuclease reaction product following the protocol (no resistance)
B. Restriction endonuclease reaction product following the protocol (with resistance)
C. 40 μL system restriction endonuclease reaction product
D. 40 μL system restriction endonuclease reaction product
E. Plasmid transformation from day 7-18
B. Restriction endonuclease reaction product following the protocol (with resistance)
C. 40 μL system restriction endonuclease reaction product
D. 40 μL system restriction endonuclease reaction product
E. Plasmid transformation from day 7-18
2021-7-21
- No conoly from day 7-20 (ABCDE)
- DNA agarose gel electrophoresis identification of day 7-20 DNA connection
reaction, found that there’s no stripes (very light)
- Clip connection
- Plasmid transformation (change from 10min RTP to 20 Celsius 1h)
F. E. coli without transfer in plasmid in the non-resistant LB medium to make sure E. Coli sill
working
G. Transfer in Pprovoin5 plasmid
H. Plasmid transformation from 7-21
I. Plasmid transformation from 7-21
J. Plasmid transformation from 7-21
G. Transfer in Pprovoin5 plasmid
H. Plasmid transformation from 7-21
I. Plasmid transformation from 7-21
J. Plasmid transformation from 7-21
2021-7-22
- No conoly until afternoon second one from 7-21 has some colony, so plasmid
extraction 16h amplification
- Restriction endonuclease reaction
• Leave it in 37 celsius
through night
- DNA clip connection reaction (increase the amount of plasmid and leave 16 Celsius
through night)
• AmiGFP: Pprovoin5 = 4.5 μL :
1.5 μL
2021-7-23
- Plasmid extraction from day 7-22 amplification
• So we could have more
Pprovoin5 plasmid
- DNA agarose gel electrophoresis identification
• Marker changed
- 5 μL marker
- 0 μL loading buffer
- 2 μL Nucleic acid dyestuff
- 0 μL loading buffer
- 2 μL Nucleic acid dyestuff
• Negative control (only amiGFP
plasmid because we don’t have enough Pprovoin5 plasmid)
2021-9
- Repeat the plasmid B constrcution
- Coloy PCR to verify the transformants of plasmid B and extract the plasmid for sequencing
- Prepare LB medium with two antibiotics (kana&)
- Extract the plasmid A and plasmid B to be transformed into DH5a to obtain bacteria C
- Coloy PCR to verify the transformants of plasmid B and extract the plasmid for sequencing
- Prepare LB medium with two antibiotics (kana&)
- Extract the plasmid A and plasmid B to be transformed into DH5a to obtain bacteria C
2021-10-1~7
- Conduct function tests
- Cell-free extraction experiments
