Notebook
2019/7/16
Content of the experiment: Colony PCR
Raw material: LB medium
chloramphenicol
Colony of bacteria
Steps:
1. Take 2 mL LB culture medium and add it into two 2 mL EP tubes.
2. 2 uL chloramphenicol was added to the EP tubes.
3. Blend slightly.
4. Take 15 1.5mL EP tubes.
5. 200 uL medium containing anti-LB was added into EP tube.
6. Five colonies were taken from each Petri dish and added into the corresponding EP tube.
7. Shake bacteria on shaking bed at 37 C for 2 hours.
Result:
The turbidity of the bacterial liquid indicates that the bacterial body has successfully reproduced and the concentration of the bacterial liquid is high.
Tips:
1. We made three strains, s, W and m.
2. Chloramphenicol is a screening agent, which can remove impurities.
2019/7/18
Content of the experiment: Measuring OD value
Raw materials:
Eon enzyme labelling instrument
Diluted S4 W1 M5
S4 W1 M5 stock solution
S3 M2 W2 stock solution
Steps: (For detailed steps of multiple dilution, see the relevant documents) 1. Two 1.groups of diluted S4 W1 M5 and 100ul each were added to the enzyme label plate.
2. Put the enzyme labeling plate into the enzyme labeling instrument to measure its OD value (see figure for data).
The measured OD value is about 0.05, not between the most suitable OD value (0.4-0.6), so the raw liquor with higher concentration is taken to measure OD value again.
3. The raw liquor of S4 W1 M5 and 100ul of each group were added to the enzyme label plate.
4. Put the enzyme labeling plate into the enzyme labeling instrument to measure its OD value (see figure for data).
The measured OD value is more than 0.6, slightly higher than the most suitable OD value (0.4-0.6). Therefore, W 1 with the smallest difference between the two values (the smallest error) is selected as the ratio dilution. OD value is measured again, and the functional relationship between OD and concentration is established to find the appropriate dilution multiple.
5. Dilute the ratio of raw solution of W1 to 1, 2, 4 and 8 times of concentration and add two groups of 100ul to the enzyme label plate.
6. Put the enzyme labeling plate into the enzyme labeling instrument to measure its OD value (see the yellow part of the icon for the data).
The function curve of OD value with respect to concentration is established with Excel table (see figure).
The OD value is 0.5926 when the OD value is 0.4 and 0.635 when the concentration multiple is 1, so the dilution multiple should be 1/0.5926 = 1.6874 for the original solution of W1. (principle of calculation: concentration = volume / volume, concentration is inversely proportional to volume, and dilution multiple is equal to volume multiplier, so the concentration ratio is inversely proportional to dilution multiple, and the concentration multiple is the reciprocal of dilution multiple).
Because the raw liquor of S4, W1 and M5 is not much surplus, it is decided to take S3, W2 and M2, and to measure the required dilution multiples for use in subsequent inspection.
7. Two groups of 100ul each of S3 W2 M2 bacterial solution were added to the enzyme label plate.
8.Put the enzyme labeling plate into the enzyme labeling instrument to measure its OD value (see figure for data).
The measured OD value is about 0.7, slightly higher than the most suitable OD value (0.4-0.6), so the average values of S3, W2 and M2 are taken into the function system.
9. Calculate the required dilution multiples:
Result:
According to the calculation results, it can be seen that the dilution factor is about 2 times.
2019/7/19
Content of the experiment: Observation of bacteria under ultraviolet radiation
Raw materials:
Ultraviolet irradiator
Resuspended centrifuged bacteria
Steps:
The groups S, W and M were irradiated by ultraviolet instrument and photographed. Principle:
When mCherry is repressed by lead, it expresses fluorescent protein and fluoresces under ultraviolet irradiation.
Result:
Group S: Compared with the control group, S ^ 10-4 showed darker colour, while the other bacteria in lead concentration showed no obvious fluorescence.
Group M: Compared with the control group, M ^ 10-4 showed darker colour, while the other bacteria in lead concentration showed no obvious fluorescence.
Group W: Compared with the control group, the bacteria of W ^ 10-4 showed a deeper color, and the bacteria with lead concentration of 10-7 had obvious red fluorescence, while the bacteria with other lead concentration had no obvious fluorescence.
Abnormal situation and analysis:
1. W10 ^ - 7 is the only visible red fluorescence in all bacterial fluids.
- It may be that the high concentration of lead acetate (toxic) leads to the death of other strains, or that mCherry cannot express (emits red fluorescence).
- It may be that in S and M, the concentration of PbrR is too high, or the repression ability is too strong, which results in the absence of mCherry expression in bacterial solution.
- It may be that the system of W10 ^ - 7 has unexpected abnormalities (mutation of strain (base deletion) or changes in expression.
2. S ^ 10-4, M ^ 10-4, W ^ 10-4 are darker and some are black.
- It may be that high lead concentration leads to cell death.
2019/7/23
Content of the experiment: Enzyme linkage
Raw materials:
TaKaRa: DNA Ligation Kit Ver.2.1
Solution I
Enzymatic digestion products of low-copy skeleton
Enzymatic Digestion Products of Synthetic Fragments of pPbrR-S4 and mCherry Enzymatic products of the synthetic fragments of pbrr-m5 and mcurry
Enzymatic Digestion Products of Synthetic Fragments of pPbrR-W1 and mCherry DdH2O
Steps:
1. Take 4 PCR tubes
2. Solution I with 4ul each
3. Low-copy skeletons with 1 UL each
4. The synthetic fragments of pPbrR-S4 and mCherry, pPbrR-M5 and mCherry, pPbrR-W1 and mCherry were added respectively.
5. Add 1 UL of ddH_2O each.
6. Set the temperature of 16 degrees Celsius in the PCR for 1 hour. Tips: After the connection, it needs to be instantly transformed into a receptive state.
2019/7/23
Content of the experiment: Axygene
Raw materials:
Agarose Gel Containing Purpose DNA
Buffer DE-A 400 UL
Buffer DE-B 200 ugl
Buffer W1 500 ugl
Buffer W2 700 mu l*2 = 1400 Mu L
Eluent 25 UL
Instruments:
blb
2.0 ml centrifugal tube
1.5 ml centrifugal tube
Water bath
DNA preparation tube
Centrifuge
Refrigerator
Steps:
1. Cut agarose gel containing target DNA under purple lamp and place it in 2.0 ml centrifugal tube. -
2. Add 400 ml Buffer DE-A, mix evenly, then heat in 75 C water bath, mix in intervals (every 2-3 minutes) until gel block melts completely (5 minutes).
3. Add 200 ml Buffer DE-B and mix evenly.
4. Absorbing the mixture of the above steps and transferring it to the DNA preparation tube (placed in 2 ml centrifugal tube), centrifuging for 1 minute at 12 000*g, discarding the filtrate
5. Put the prepared tube back into centrifugal tube, add 500 ml buffer W1, 12 000*g centrifugal for 30 seconds, discard filtrate
6. he prepared tube was placed back into the centrifugal tube, adding 700 ml buffer W2, 12 000*g centrifugal for 30 seconds, discarding the filtrate. Wash again in the same way with 700 ml buffer W2 and centrifuge for 1 minute at 12000*g.
7. Place the tube in a 2 ml centrifugal tube and centrifuge for 1 min at 12000*g. - The preparation tube was placed in a clean 1.5 ml centrifugal tube. 25 ml Eluent was added in the center of the DNA preparation membrane. The tube was placed at room temperature for 1 minute and centrifuged at 12 000*g for 1 minute to elute DNA.
8. The recovered DNA samples were stored at 4 C for short-term storage for subsequent experiments.
Tips
- Waterbaths should be intermittently checked for gel melting
- When using centrifuges, attention should be paid to the symmetrical placement of centrifugal tubes. If there are odd number of centrifugal tubes, balance of the total mass of the symmetrical centrifugal tubes (i.e. the sum of the mass of the centrifugal tubes and the mass of the substances therein) should be used.
2019/7/23
Content of the experiment: Purification and validation
Steps:
1. Place the plywood in the electrophoresis tank and add the electrophoresis buffer.
2. Adding 4 UL marker to an end-point sample hole
3. The purified products were added to 1 UL Loading Buffer with 2 UL each.
4. The purified products were added into the dot-like pore in the order of vector, S, M and W (when the black band was near the observer) from left to right.
5. Adding 4 ml marker to the end point sample hole
6. Open the electrophoresis apparatus and adjust the voltage to 90V to make the nucleic acid sample swim towards the positive pole (note that the sample hole is at the negative end of the electrophoresis tank). Time: 40 minutes
After the electrophoresis was completed, the power supply was cut off, the gel was taken out, the electrophoresis results were observed on the ultraviolet transmitter and photographic records were taken.
Tips:
1. Set the alarm clock to remind you to check the progress of running glue at intervals and stop running glue when you reach the total progress of 2/3 from the beginning.
Vector, M, S all run out of the bright band
W did not run out of the bright band.
Anomaly analysis:
It may be that the concentration of W bacterial solution is too low, which leads to the inadequate appearance of bright bands in the proposed plasmid concentration.
2019/7/23
Experiments: Enzymatic digestion of rubbing gum
Steps:
7. Place the plywood in the electrophoresis tank and add the electrophoresis buffer.
8. Adding 4 UL marker to the end-point sample hole
9. Put 50 ml of enzymatic digestion products from left to right in the order of Vector, S, M, W (when the black band is close to the observer) and then add them into the dot-like pore.
10. Add 4 ml marker to the end point sample hole
11. Open the electrophoresis apparatus and adjust the voltage to 90V to make the nucleic acid sample swim towards the positive pole (note that the sample hole is at the negative end of the electrophoresis tank).
Time: 70 minutes
- After the electrophoresis was completed, the power supply was cut off, the gel was taken out, the electrophoresis results were observed on the ultraviolet transmitter and photographic records were taken.
Tips:
2. Set the alarm clock to remind you to check the progress of running glue at intervals and stop running glue when the target strip reaches about 1/2 of the total length of the glue.
S, M and W run out of 3000, 2000 and 1500 BP bands respectively.
Vector runs out a bright band of about 2000 bp.
Analysis of abnormal results:
S, M and W run out of the bright band of about 3000bp
1. It may be that the short cleavage time resulted in the failure of some S, M and W plasmids to be cut off.
2019/7/23
Plasmid extraction:
Raw materials:
1 Bottle of Bacterial Solution: W1
Test tube, liquid gun, centrifuge, oscillator;
Kit: Hlingene High Purity Plasmid Small Quantity Rapid Extraction Kit, Shanghai Huiling Biotechnology Co., Ltd.
Buffer (Protecting DNA, Resuspending DNA), S2 (Alkaline lysis), S3 (Acidic Neutralizer + Protein Precipitation Promotion), washer 1, Eluent.
Step report:
Collection centrifugation: 1.5 ml centrifugal tube with 1.3 ml bacterial solution, 12 000 rpm centrifugation for 30 seconds, discarding supernatant
Repeat the above process 4 times and add 5.2 ml bacterial solution.
Resuspension: adding 500 UL buffer S1, the shock is sufficient to completely free of small bacteria;
Move all solutions to 2ml EP tube
Pyrolysis: Add 500 UL of S 2, gently inverted about 10 times, so that the solution becomes clear; Neutralization: adding 700ul S3, gently inverted about 10 times, white flocculent objects appeared, centrifuged at 13000 rpm for 15 minutes;
Collection: absorb supernatant (do not touch white precipitation) 600ul, transfer it into the adsorption column of the collection tube, centrifuge at 12000 rpm for 30 seconds, and pour out the waste liquid; repeat the above action once
Cleaning: add 600ul WB lotion to the adsorption column, centrifuge at 12000rpm for 30s, discard the filtrate, and repeat it.
Air centrifugation: 12000 RPM centrifugation for 30 seconds;
The column was replaced by a new 2ml centrifugal tube.
Elution of the heart: add 65ul (50-100ul) eluent, stand for 2 minutes, centrifuge for 1 minute at 12000 rpm.
Tips:
1. New collecting tubes need to be replaced from centrifugation to heart washing. 2. When adding bacterial liquid, it is necessary to see the mark clearly, and the pipes need to be corresponded.
3. Use up the solution and remember to cover it.
4. The eluent needs to be preheated in advance.
Concentration 132.5
Abnormal:
1. The centrifuged S and M are reddish, the reason is unknown.
2019/7/24
Experiment content: enzyme cutting
Pbrr-s 50ul system：
10x green buffer 5ul
EcoRI 2ul
PstI 2ul
Ppbrr-s 14ul
DdH2O 27ul
Pbrr-m 50ul system：
10x green buffer 5ul
EcoRI 2ul
PstI 2ul
Ppbrr-m 22ul
DdH2O 19ul
Pbrr-w 50ul system：
10x green buffer 5ul
EcoRI 2ul
PstI 2ul
Ppbrr-w 41ul (41.9ul should have been added)
DdH2O 0ul
Low copy skeleton vector 50ul system：
10x green buffer 5ul
EcoRI 2ul
PstI 2ul
Skeleton plasmid 14ul
DdH2O 27ul
step
1. Match the above systems
2. Put in the PCR instrument, 37 ℃, 45 minutes
Tips:
1. Enzyme should be added first
2019/7/24
Experiment content: PCR
20ul system
1. Taq mix (2x) 10ul
2. Primer (F1) 1ul
3. Primer (R2) 1ul
4. Template (bacterial solution) 1ul
5. DdH2O 7ul
PCR process:
Set 20ul
Start:
94 ℃ 5min
Cycle step (34 times):
94 ℃ 30s
55 ℃ 30s
72 ℃ 1min 40s
End:
72 ℃ 5min
Finally placed at 4 ℃
Raw materials:
Taq 2x Mix
Up primer F1
Lower primer R2
bacterial fluid
DdH2O
Steps:
1. Mark 16 PCR tubes
2. Add 10ul Taq enzyme solution to each PCR tube.
3. Add 7ul ddH2O into each PCR tube
4. Add 1ul F1 primer to each PCR tube
5. Add 1ul R2 primer to each PCR tube
6. Add 1ul s bacteria solution to 5 tubes, 1ul m bacteria solution to 5 tubes, 1ul w bacteria solution to 5 tubes, and 1ul ddH2O to the remaining tube.
7. Put all configuration products into PCR machine
8. Set up PCR instrument:
Set volume to 20ul
The pre denaturation temperature is 94 ℃, 5 minutes
Repeat the following three steps 34 times
Denaturation temperature 94 ℃, 30 seconds
Annealing temperature 55 ℃, 30 seconds
Extension temperature 72 ℃, 1 minute 40 seconds
Set to stay at 72 ℃ for 5 minutes
Last stop at 4 ℃
Tips:
1. When matching the PCR system, it is necessary to confirm whether the measurement and proportion are accurate. If bubbles appear, it is necessary to take them again.
2. When adding bacterial solution to PCR tube, it is necessary to confirm whether it corresponds to
3. Pay attention to the accuracy of input value when setting up PCR instrument
1. S1 did not run out of bright band, S2, S3, S4, S5 all ran out of bright band.
2. M3 and M4 did not run out of bright band, M1, M2 and M5 all ran out of bright band.
3. W ran out of the bright band.
(Abnormal) There is a dark band in the negative control, the reason is unknown.
2019/7/24
Experiment content: EC20 2 SMTA 2
raw material
LB liquid medium
EC20 2 bacteria solution
SmtA 2 bacteria solution
Experimental steps
EC20 2
-20 ml LB medium was added into a large glass bottle.
-Add 40 μ L acellin to the glass bottle, and mark ec202 on the bottle body.
-Add 80 μ L ec202 bacterial solution into the glass bottle.
SmtA
-5 ml of LB medium was added to the other four glass bottles, and SMTA 2 was labeled on the bottle body.
-Add 10 μ l of acellin into the glass bottle.
-Add 20 UL of SMTA 2 solution to the glass bottle.
-Put all glass bottles into a 37 ℃ shaker for cultivation
2019/7/24
Experiment content: amplification of SL ml WL
raw material
LB liquid medium
SL bacteria solution
ML bacteria solution
WL bacteria solution
Experimental steps
-4 ml LB medium was added into 15 glass bottles.
-Add 8ul acellin to 15 glass bottles.
-Add 10 μ l of bacteria solution containing s into five glass bottles and mark SL on the bottle body
-Add 10 μ l of bacteria solution containing m into the other five glass bottles and mark ml on the bottle body
-Add 10 μ l of W bacteria solution into the last five glass bottles, and mark WL on the bottle body.
-Put all glass bottles into a 37 ℃ shaker for cultivation
2019/7/25
Experiment content: amplified bacterial solution S2 M1 W3
raw material
LB liquid medium
S2 bacteria solution
M1 bacteria solution
W3 bacteria solution
Ampicillin
Experimental steps
-10 ml LB medium was added into three glass bottles.
-Add 30ul of acellin into 3 glass bottles respectively.
-Add 2ml of S2 bacteria solution into a glass bottle, and mark S2 l 7.25 on the bottle body.
-Add 2ml of M1 bacteria solution into another glass bottle, and mark M1 l 7.25 on the bottle body.
-Add 2ml of W3 bacteria solution into the last glass bottle, and mark w3l 7.25 on the bottle body.
-Put all glass bottles into a 37 ℃ shaker for cultivation
2019/7/25
Experiment content: enzyme cutting and glue releasing
Steps:
-Put the rubber plate into the electrophoresis tank and add the electrophoresis buffer.
- Add 4 μ l marker into the sample hole at one end
-Add 10 μ l enzyme products from left to right in the order of W5, W3, W4, M2, M1, S5, S4, S2 (when the black strip is close to the observer) into the sample hole in turn.
-Add 4 μ l marker to the end sample hole
- Turn on the electrophoresis instrument, adjust the voltage to 90V, and make the nucleic acid sample swim towards the positive pole (note that the sample hole is at the negative end of the electrophoresis tank). Time: 70 minutes
-After electrophoresis, the power is cut off, the gel is removed, and the electrophoresis results are recorded on the ultraviolet transmittance instrument and recorded.
Tips:
3. Set alarm clock to remind, check the glue running progress according to the interval, and stop the glue running when the target strip reaches about 1 / 2 of the total glue length.
S, m and w all run out of two bright bands
One is about 2000 BP and one is about 1500 bp.
In line with the ideal situation.
2019/7/25
Experiment content: enzyme cutting
20ul system of S (S2 S4 S5), m (M1 m2), w (W3 W4 W5) plasmids
10x green buffer 2ul
EcoRI 1ul
PstI 1ul
DNA 5ul
DdH2O 11UL
step
-Match the above systems
2019/7/25
Experiment content: Plasmid Extraction:
Raw materials:
8 bottles of bacterial solution: S2, S4, S5 M1, M2 W3, W4, W5
Tube, liquid gun, centrifuge, shaker;
Kit: Shanghai Huiling Biotechnology Co., Ltd. hlingene high purity plasmid small quantity rapid extraction kit
Buffer, S2, S3, washer1, eluent.
Step report:
Collection and centrifugation: add 1.3ml bacterial solution into 2ml centrifuge tube, centrifugate at 12000rpm for 30s, and discard the supernatant.
Repeat the above process for three times, and add 3.9ml bacterial solution in total. Heavy suspension: add 250ul of buffer S1, shake fully until there is no small bacteria block;
Cracking: add 250ul S2, turn it upside down gently for about 10 times to make the solution clear;
Neutralization: add 350 UL S3, turn it upside down gently for about 10 times, white floccules appear, centrifugation at 13000 rpm for 10 minutes;
Collection: take 600ul of supernatant (do not touch the white precipitate), transfer it into the adsorption column of the collection tube, centrifugate it at 12000rpm for 30s, and pour out the waste liquid;
Washing: add 600ul WB washing solution to the adsorption column, centrifugate at 12000rpm for 30s, and discard the filtrate; repeat once
Air centrifugation: 13000rpm centrifugation for 2min;
Replace the column with a new 2ml centrifuge tube.
Elution centrifugation: add 65ul (50-100ul) eluent, stand for 2min, centrifugation at 12000rpm for 1min.
Tips:
-A new collecting tube is needed during the process from air centrifugation to elution centrifugation.
-When adding the bacterial solution, it is necessary to see the mark clearly, and the added pipes need to be corresponding.
-Remember to cover the used solution.
-The eluate needs to be preheated in advance.
According to 260 / 280 (purity) value
We finally selected S2, M1 and W3 for sequence detection.
2019/7/29
Experiment content: amplification of bacterial solution S4 M2 W5
raw material
LB liquid medium
S4 bacteria solution
M2 bacteria solution
W5 bacteria solution
Ampicillin
Experimental steps
-4 ml LB medium was added into 3 plastic tubes.
-8 UL acellin was added into 3 plastic tubes respectively.
-Add 20ul of S4 bacterial solution into a plastic tube and mark the bottle with S4 l 7.28.
-Add 20ul of M2 bacteria solution into another plastic tube, and mark M2 l 7.28 on the bottle body.
-Add 20ul of W5 bacteria solution into the last plastic tube, and mark W5 l 7.28 on the bottle body.
-Put all plastic tubes into a 37 ℃ shaker for cultivation
July 30, 31, 2019
Experiment content: measuring the growth curve of bacteria
Raw materials:
LB medium
Ampicillin
S4L bacteria solution
M2L bacteria solution
W2L bacteria solution
Lead acetate solution with multiple dilution
Steps:
1. Add 5ml LB medium into 6 plastic tubes respectively
2. Add 10ul acellin into these plastic pipes respectively.
3. Add 50ul s4l bacterial solution into these plastic tubes respectively.
4. In the first tube, add 5ul 10 ^ 1 lead acetate solution with the standard of s4l 10 ^ 1 7.30.
5. Add 5ul 10 ^ 2 lead acetate solution in the second tube, and the standard is s4l 10 ^ 2 7.30.
6. In the third tube, add 5ul 10 ^ 3 lead acetate solution with the standard of s4l 10 ^ 3 7.30.
7. Add 5ul 10 ^ 4 lead acetate solution in the fourth tube, and the standard is s4l 10 ^ 4 7.30.
8. Add 5ul 10 ^ 5 lead acetate solution in the fifth tube, marked as s4l 10 ^ 5 7.30. 9. No lead acetate solution is added in the sixth tube, and the standard is s4l 0 7.30. 10. Take 400ul s4l 10 ^ 1 7.30 solution and add it into 1.5ml EP tube, which is marked as s 10 ^ 1 0h 7.30.
11. Take 400ul s4l 10 ^ 2 7.30 solution and add it into 1.5ml EP tube, which is marked as s 10 ^ 2 0h 7.30.
12. Take 400ul s4l 10 ^ 3 7.30 solution and add it into 1.5ml EP tube, which is marked as s 10 ^ 3 0h 7.30.
13. Take 400ul s4l 10 ^ 4 7.30 solution and add it into 1.5ml EP tube, which is marked as s 10 ^ 4 0h 7.30.
14. Take 400ul s4l 10 ^ 5 7.30 solution and add it into 1.5ml EP tube, which is marked as s 10 ^ 5 0h 7.30.
15. Take 400ul s4l 0 7.30 solution, add it into 1.5ml EP tube, and mark it as s00h 7.30. 16. Put all EP pipes into 4 ℃ for storage.
17. Repeat the above steps for m2l and w5l.
18. After that, 400ul of each plastic pipe shall be sampled every 2 hours and put into 1.5ml EP pipe and marked accordingly (for example, the sample with the date of 7.30 on the same day and swaying for 2h in s4l 10 ^ 1 shall be marked with s 10 ^ 1 2H 7.30) until 8 hours.
19. Take three 100ul samples from all samples and add them into the enzyme standard plate to measure their OD value.
According to the OD value, the growth curve of bacteria was drawn under different lead concentration.