Team:Heidelberg/Experiments
Methods
Me pipette? Yeah. Me contaminate? No.
Normally, gels were used with a concentration of 1 % agarose. However, for really small DNA fragments and for the gel extraction, gels with an agarose concentration of 2 % were used.
The following protocol was followed:
The agarose was dissolved in 1x TAE buffer by boiling. Per 100 ml agarose solution 5 µl ethidium bromide was added.
The gel was poured into a gel electrophoresis chamber and it was waited until it completely cooled down. Then it was placed into the tank and it was filled up with 1x TAE buffer until the whole gel was covered.
0.5 µl of a 6x purple loading dye were mixed with 2.5 µl of the sample. The mixtures were loaded into the gel pockets.
As a ladder, the DirectLoad Wide Range DNA Marker was used.
The gel was run at 90 V for around 40 minutes and imaged using a UV transilluminator.
The following protocol was followed:
The following buffers were prepared before beginning:
100 mM MgCl2, 100 mM CaCl2 and 85 mM CaCl2, 15 % glycerol
The buffers without glycerol were autoclaved. The buffer with glycerol was sterile filtered with a 0.22 µm filter.
The following protocol was followed to prepare the competent Top 10 cells:
10 ml LB medium was inoculated with the Top 10 strain (as a friendly gift of the AG Niopek) and incubated overnight at 37 °C and 225 rpm.
On the next day, two flasks of 500 ml LB medium were each inoculated with 5 ml of the overnight culture. It was grown to an OD600 of 0.35 - 0.4.
The cells were cooled for 30 minutes on ice. From this step on, the cells were kept on ice.
The cells were split into 20 pre-cooled 50 ml falcon tubes and centrifuged for 15 minutes at 3000 xg. The supernatant was discarded.
The pellet was resuspended in 20 ml of pre-cooled MgCl2. The mixture was centrifuged for 15 minutes at 2000 xg. Again, the supernatant was discarded.
The pellet was resuspended in 10 ml of pre-cooled CaCl2. The content of five falcon tubes was combined and chilled for 20 minutes on ice. Again, it was centrifuged for 15 minutes at 2000 xg.
The pellet was resuspended in 12.5 ml pre-cooled 85 mM CaCl2, 15 % glycerol. The content of the falcons was combined and centrifuged for 15 minutes at 2000 xg.
The pellet was resuspended in 2 ml pre-cooled 85 mM CaCl2, 15 % glycerol. Each 50 µl was aliquoted into pre-cooled Eppendorf tubes. The aliquots were snap-frozen in liquid nitrogen and stored at -80 °C.
Small amounts of the cells were spread on agar plates with resistance genes, to check whether the cells were contaminated with a resistance gene.
The buffers without glycerol were autoclaved. The buffer with glycerol was sterile filtered with a 0.22 µm filter.
The following protocol was followed to prepare the competent Top 10 cells:
For the fluorescence measurement in the plate reader, the following protocol was followed:
Competent Top 10 cells were transformed with Level 1 broccoli plasmids and grown on an agar plate.
Five colonies were picked per plate and an overnight seed culture prepared. Another seed culture is prepared with cells not transformed with a plasmid. This culture is referred to as crude culture.
On the next day, 3 ml of the seed cultures were diluted to an OD 600 of 0.4 in LB medium containing the appropriate antibiotics.
The dilution was incubated for two to four hours.
A buffer of 40 µM DFHBI in PBS was prepared. DFHBI was diluted beforehand in DMSO to a concentration of 40 mM.
1 ml of the buffer was mixed with 20 µl of the seed culture and incubated for 30 minutes.
Two negative controls were prepared. One negative control was prepared with 20 µl LB medium and one with 20 µl of the crude culture, both mixed with 1 ml of the DFHBI-PBS buffer. The negative controls were also incubated for 30 minutes.
The OD600 of all cultures and negative controls was measured.
100 µl of each culture and negative control were pipetted in wells of a black 96-well plate. Like this of each culture four replicates were made.
The fluorescence intensity was measured with a plate reader at an excitation maximum of 447 nm and an emission maximum of 501 nm.
For analysis the means of each replicate pairs were calculated. The background fluorescence (fluorescence intensities of PBS and 40 µM DFHBI medium without bacteria) were substracted. All fluorescence intensities were normalised to equal bacterial concentrations by dividing the values through the OD600 that was measured earlier. Finally, the mean values of the seed cultures of the broccoli colonies were divided by the mean value of the crude seed culture to compare the fluorescent intensity of the probes to the negative control.
For the measurement of fluorescent proteins, the following protocol was followed:
Competent Top 10 cells were transformed with the appropriate plasmids to obtain colonies with the correct probes. The bacteria was grown on an agar plate overnight.
Five colonies were picked per plate and an overnight seed culture was prepared. Another seed culture is prepared with cells not transformed with a plasmid. This culture is referred to as crude culture.
On the next day, 3 ml of the seed cultures were diluted to an OD 600 of 0.4 in LB medium with the respective antibiotic.
It was incubated for two to four hours.
Two negative controls were prepared. One negative control was prepared with LB medium and one with the crude culture.
The OD of all cultures and negative controls was measured.
100 µl of each culture and negative control were pipetted in wells of a black 96-well plate. Like this of each culture four replicates were made.
The fluorescence intensity was measured with a plate reader at an excitation maximum of 485 nm and an emission maximum of 510 nm in the case of sfGFP, at an excitation maximum of 468 nm and an emmision maximum of 505 nm in the case of assembled split-GFP or at an excitation maximum of 569 nm and emmission maximum of 594 nm in the case of mScarlet.
For analysis the means of each replicate pairs were calculated. The background fluorescence (fluorescence intensities of LB medium without bacteria) were substracted. All fluorescence intensities were normalised to equal bacterial concentrations by dividing the values through the OD600 that was measured earlier. Finally, the mean values of the probes were divided by the mean value of the crude seed culture, to compare the fluorescent intensities of the probes to the negative control
Gel Extractions were performed with the NEB Monarch Gel Extraction Kit as proposed by the manufacturer. For gel extraction, a higher volume of the PCR reaction mixture was prepared. 100 µl of the PCR product was loaded on a gel. For getting big enough pockets in the gel, tape was clued around two tooths of a gel comb.
1 ml of an overnight culture were transferred in an Eppendorf tube and centrifuged at 2500 rpm for 15 minutes. The supernatant was decanted and the pellet was resuspended in 1 ml LB with 20 % glycerol. The glycerol stocks were sorted into freezer boxes and stored in the -80°C freezer.
Level 0 Golden Gate Cloning was done with the following components:
75 ng PCR product (as the concentration could not be measured we used 2 µl)
7.5 ng entry vector
1 µl T4 DNA Ligase buffer
1 µl T4 DNA Ligase or Hi-T4 DNA Ligase
0.5 µl EspI3
Milli-Q water to 10 µl
In the thermocycler the following program was followed:
After the cloning, an analytical gel electrophoresis was performed.
Table 1: Thermocycler settings for Golden Gate Level 0 cloning. For the T4-Ligase the ligation temperature in Step 2 was changed to 16 °C.
| Step | |
|---|---|
| Step 1 | 37 °C, 1 min 30 sec |
| Step 2 | 25 °C, 3 min |
| Repetition of Step 1 and 2 | 15 x |
| Step 3 | 50 °C, 5 min |
| Step 4 | 80 °C, 10 min |
| Step 5 | 4 °C, until lid is opened |
Level 1 Golden Gate Cloning was done with the following components:
75 ng of each DNA insert
1 µl T4 DNA Ligase buffer
1 µl T4 DNA Ligase or Hi-T4 DNA Ligase
0.5 µl Bsa3I
Milli-Q water to 10 µl
In the thermocycler the following program was followed:
Level 1 cloning was performed for plasmids gaining three different antibiotic resistances: Ampicillin-resistance, Kanamycin-resistance, Tetracycline-resistance.
After the cloning, an analytical gel electrophoresis was performed.
Table 2: Thermocycler settings for Golden Gate Level 1 cloning. For the T4-Ligase the ligation temperature in Step 2 was changed to 16 °C.
| Step | |
|---|---|
| Step 1 | 37 °C, 2 min |
| Step 2 | 25 °C, 5 min |
| Repetition of Step 1 and 2 | 50 x |
| Step 3 | 50 °C, 10 min |
| Step 4 | 80 °C, 10 min |
| Step 5 | 4 °C, until lid is opened |
Level 1 cloning was performed for plasmids gaining three different antibiotic resistances: Ampicillin-resistance, Kanamycin-resistance, Tetracycline-resistance.
After the cloning, an analytical gel electrophoresis was performed.
Level 2 Golden Gate Cloning was done with the following components:
75 ng of each DNA insert
1 µl T4 DNA Ligase buffer
1 µl T4 DNA Ligase or Hi-T4 DNA Ligase
0.5 µl EspI3
Milli-Q water to 10 µl
In the thermocycler the following program was followed:
Note: We were not able to construct a finished Level 2 cloning construct in our limited time in the lab, but would highly suspect the thermocycler conditions stated in Table 3.
After the cloning, an analytical gel electrophoresis was performed.
Table 3: Thermocycler settings for Golden Gate Level 2 cloning. For the T4-Ligase the ligation temperature in Step 3 was changed to 16 °C.
| Step | |
|---|---|
| Step 1 | 37 °C, 5 min |
| Step 2 | 25 °C, 16 min |
| Repetition of Step 1 and 2 | 30 x |
| Step 3 | 37 °C, 30 min |
| Step 4 | 80 °C, 10 min |
| Step 5 | 4 °C, until lid is opened |
After the cloning, an analytical gel electrophoresis was performed.
For example, the Pumbys had to be synthesized in two separate parts. To combine them, a digest with BsaI and a ligation were performed. The following components were combined, mixed by pipetting, and put in the thermocycler, following the program: incubate for 60 min (37 °C), deactivate 10 min (65 °C), stay at 4°C.
5 µl of Pumby C-terminal and N-terminal PCR products
2.5 µl T4 DNA Ligase Buffer 10X
1.2 µl T4 DNA Ligase (500 U)
0.75 µl BsaI (15 U)
10.55 µl Milli-Q water
The minipreps were conducted with the PureYield Plasmid Miniprep System of Promega as proposed by the manufacturer. The alternative protocol for larger culture volumes was amended. At first, 1.5 ml of bacterial culture were centrifuged at 10 000 rpm for 30 seconds. The supernatant was discarded and another 900 µl of the bacteria culture was added to the pellet and also centrifuged. The now resulting pellet was resuspended in another 600 µl bacterial culture.
After the miniprep, the concentration was measured with the Implen N80 Nanophotometer. As a blank, the elution buffer was used.
After the miniprep, the concentration was measured with the Implen N80 Nanophotometer. As a blank, the elution buffer was used.
Overlap extension PCRs were performed with double the amount of primer volume.
The following was combined into a PCR-Tube:
0.5 µl of each primer at a concentration of 50 µM
1 µl T4 DNA Ligase buffer
10 ng DNA template
12.5 µl PCR Mastermix (NEBNext® High-Fidelity 2X PCR Master Mix or NEB Taq 2X Master Mix)
Milli-Q water to 25 µl
In the thermocycler, the following program was used for the overlap extension PCRs:
The PCRs were verified with analytical Gel electrophoresis.
The following was combined into a PCR-Tube:
Table 4: Thermocycler settings for PCRs. The annealing temperature (Step 3) and extension time (Step 4) were determined for each primer pair with the NEB Tm calculator. For the used temperatures and times, check out our notebook.
| Step | High-Fidelity 2X PCR Master Mix | Taq 2X Master Mix |
|---|---|---|
| Step 1 (Initial Denaturation) | 98 °C, 30 sec | 95 °C, 30 sec |
| Step 2 (Denaturation) | 98 °C, 10 sec | 95 °C, 25 sec |
| Step 3 (Annealing) | variable, 20 sec | variable, 20 sec |
| Step 4 (Extension) | 72 °C, variable | 68 °C, variable |
| Repetition of Steps 2 to 4 | 30 x | 30 x |
| Step 5 (Last Extension) | 72 °C, 2 min | 68 °C, 2 min |
| Step 6 | 4 °C, until lid is opened | 4 °C, until lid is opened |
PCRs were performed with the DNA parts and primers from IDT. The following volumes were combined in a PCR tube:
0.25 µl of each primer (Reverse and Forward) at a concentration of 50 µM
1 µl T4 DNA Ligase buffer
10 ng DNA template
12.5 µl PCR Mastermix (NEBNext® High-Fidelity 2X PCR Master Mix or NEB Taq 2X Master Mix)
Milli-Q water to 25 µl
In the thermocycler, the following program was used for the PCRs:
Table 5: Thermocycler settings for PCRs. The annealing temperature (Step 3) and extension time (Step 4) were determined for each primer pair with the NEB Tm calculator. For the used temperatures and times, check out our notebook.
| Step | High-Fidelity 2X PCR Master Mix | Taq 2X Master Mix |
|---|---|---|
| Step 1 (Initial Denaturation) | 98 °C, 30 sec | 95 °C, 30 sec |
| Step 2 (Denaturation) | 98 °C, 10 sec | 95 °C, 25 sec |
| Step 3 (Annealing) | variable, 20 sec | variable, 20 sec |
| Step 4 (Extension) | 72 °C, variable | 68 °C, variable |
| Repetition of Steps 2 to 4 | 30 x | 30 x |
| Step 5 (Last Extension) | 72 °C, 2 min | 68 °C, 2 min |
| Step 6 | 4 °C, until lid is opened | 4 °C, until lid is opened |
Master mixes were prepared from water and the primers for the colony PCR. 490 µl water was mixed with 5 µl of both primers. Different primers were used for the Level 0 cloning, the Level 1 cloning with the various antibiotic resistances, and the Level 2 cloning. The primers were beforehand diluted to a concentration of 50 µM.
Before picking, Eppendorf tubes were prepared with 15 µl of the respective master mix. The colonies were picked with a small pipette tip. The pipette tip was first dipped two to three times in the prepared Eppendorf tube. Then, the pipette tip was thrown in a falcon for the overnight cultures (see Picking of colonies and overnight cultures).
Normally and if possible, five to ten colonies were picked per plate for bacteria transformed with Level 1 and Level 2 cloning constructs. Around three colonies were picked for bacteria transformed with Level 0 cloning constructs. It was determined with a UV transilluminator, which colonies were picked. White colonies were picked that did not emit a GFP signal.
To the master mix with cells, 12.5 µl PCR Mastermix (NEBNext® High-Fidelity 2X PCR Master Mix or NEB Taq 2X Master Mix) was added.
In the thermocycler, the following program was used for the colony PCR:
After the cloning, an analytical gel electrophoresis was performed.
Before picking, Eppendorf tubes were prepared with 15 µl of the respective master mix. The colonies were picked with a small pipette tip. The pipette tip was first dipped two to three times in the prepared Eppendorf tube. Then, the pipette tip was thrown in a falcon for the overnight cultures (see Picking of colonies and overnight cultures).
Normally and if possible, five to ten colonies were picked per plate for bacteria transformed with Level 1 and Level 2 cloning constructs. Around three colonies were picked for bacteria transformed with Level 0 cloning constructs. It was determined with a UV transilluminator, which colonies were picked. White colonies were picked that did not emit a GFP signal.
To the master mix with cells, 12.5 µl PCR Mastermix (NEBNext® High-Fidelity 2X PCR Master Mix or NEB Taq 2X Master Mix) was added.
In the thermocycler, the following program was used for the colony PCR:
Table 6: Thermocycler settings for colony PCRs. The annealing temperature (Step 3) depends on the used primers. For the Level 0 primers it is 53 °C, for the Level 1 ampicillin primers 46 °C and the tetracycline primers 51 °C and for the Level 2 primers 50 °C. For kanamycin-resistance genes no primers were available.
| Step | High-Fidelity 2X PCR Master Mix | Taq 2X Master Mix |
|---|---|---|
| Step 1 (Initial Denaturation) | 98 °C, 30 sec | 95 °C, 30 sec |
| Step 2 (Denaturation) | 98 °C, 10 sec | 95 °C, 25 sec |
| Step 3 (Annealing) | variable, 30 sec | variable, 30 sec |
| Step 4 (Extension) | 72 °C, 2 min | 68 °C, 2 min |
| Repetition of Steps 2 to 4 | 30 x | 30 x |
| Step 5 (Last Extension) | 72 °C, 5 min | 68 °C, 5 min |
| Step 6 | 4 °C, until lid is opened | 4 °C, until lid is opened |
A falcon was prepared with 4 ml LB medium inoculated with 4 µl of the preferred antibiotic. The antibiotics were used in the following concentrations: Ampicillin 100 µg/ml, Chloramphenicol 34 µg/ml, Kanamycin 30 µg/ml, Tetracycline 5 µg/ml.
The colonies were picked with a small pipette tip. The pipette tip was first dipped two to three times in an Eppendorf tube for the Colony PCR (see Picking of colonies and Colony PCR). Then, the pipette tip was thrown in the prepared falcon. The falcon was incubated at 37 °C and 225 rpm overnight.
Normally and if possible, five to ten colonies were picked per plate for bacteria transformed with Level 1 and Level 2 cloning constructs. Around three colonies were picked for bacteria transformed with Level 0 cloning constructs. It was determined with a UV transilluminator, which colonies were picked. White colonies were picked that did not emit a GFP signal.
The colonies were picked with a small pipette tip. The pipette tip was first dipped two to three times in an Eppendorf tube for the Colony PCR (see Picking of colonies and Colony PCR). Then, the pipette tip was thrown in the prepared falcon. The falcon was incubated at 37 °C and 225 rpm overnight.
Normally and if possible, five to ten colonies were picked per plate for bacteria transformed with Level 1 and Level 2 cloning constructs. Around three colonies were picked for bacteria transformed with Level 0 cloning constructs. It was determined with a UV transilluminator, which colonies were picked. White colonies were picked that did not emit a GFP signal.
The autoclaved LB agar was heated in the microwave until it was a clear liquid. It then was cooled until its temperature was approximately 55°C. The preferred antibiotics were added in the following concentrations: Ampicillin 100 µg/ml, Chloramphenicol 34 µg/ml, Kanamycin 30 µg/ml, Tetracycline 5 µg/ml.
If two antibiotics were combined, the same concentrations were used.
The plates were marked with the following system and stored in the 4°C fridge.
0 Strokes: without antibiotics
1 Stroke: Ampicillin
2 Strokes: Chloramphenicol
3 Strokes: Kanamycin
4 Strokes: Tetracycline
In the case that the agar plate had two antibiotics, it was marked with the corresponding number of strokes from the two antibiotics with a gap in between.
Before usage, the plates were warmed up on the bench for at least 15 minutes.
If two antibiotics were combined, the same concentrations were used.
The plates were marked with the following system and stored in the 4°C fridge.
Before usage, the plates were warmed up on the bench for at least 15 minutes.
The antibiotics were prepared in the following dilutions: Ampicillin 100 mg/ml, Chloramphenicol 34 mg/ml, Kanamycin 30 mg/ml, Tetracycline 5 mg/ml.
Ampicillin and Kanamycin were diluted in Milli-Q water and were filter sterilized. Chloramphenicol and Tetracycline were diluted in 99.8 % ethanol.
Ampicillin and Kanamycin were diluted in Milli-Q water and were filter sterilized. Chloramphenicol and Tetracycline were diluted in 99.8 % ethanol.
For the LB liquid medium, 1 % Bacto-Tryptone, 0.5 % Bacto-Yeast Extract, and 0.17 M NaCl were mixed and autoclaved.
For the LB agar, the LB medium was mixed with 1.5 % agar and autoclaved. The LB agar was heated until it was a clear liquid. It was waited until the liquid cooled down to around 55 °C. The preferred antibiotics were added in the following concentrations: Ampicillin 100 µg/ml, Chloramphenicol 34 µg/ml, Kanamycin 30 µg/ml, Tetracycline 5 µg/ml.
For the LB agar, the LB medium was mixed with 1.5 % agar and autoclaved. The LB agar was heated until it was a clear liquid. It was waited until the liquid cooled down to around 55 °C. The preferred antibiotics were added in the following concentrations: Ampicillin 100 µg/ml, Chloramphenicol 34 µg/ml, Kanamycin 30 µg/ml, Tetracycline 5 µg/ml.
First SOB (Super Optimal Broth) Medium was prepared from 2 % Bacto-Tryptone, 0.5 % Bacto-Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4 and autoclaved.
100 ml of the autoclaved SOB medium were mixed with a filter-sterilized 20 % glucose solution. The glucose solution was filtered with a 0.22 µm filter.
100 ml of the autoclaved SOB medium were mixed with a filter-sterilized 20 % glucose solution. The glucose solution was filtered with a 0.22 µm filter.
The DNA parts and primers which were synthesized by IDT were shipped dry. They were resuspended in the following way: The tubes were centrifuged for 5 s, Milli-Q water was added to reach a concentration of 10 ng/µl and the tubes were vortexed briefly. Afterward, they were incubated 15 minutes at 50 °C, vortexed briefly, and spun down. The resuspended DNA was stored in the -20 °C freezer.
The primers were resuspended in the following way: The tubes were centrifuged for 5 s, Milli-Q water was added to reach a 50µM solution (20 times the oligo yield in nmol) and the tubes were vortexed briefly and spun down. The resuspended DNA was stored in the -20 °C freezer.
The primers were resuspended in the following way: The tubes were centrifuged for 5 s, Milli-Q water was added to reach a 50µM solution (20 times the oligo yield in nmol) and the tubes were vortexed briefly and spun down. The resuspended DNA was stored in the -20 °C freezer.
From the Minipreps we prepared, we took samples and prepared them for sequencing in Economy Sanger Sequences from Microsynth. The probes were prepared after the directions of Microsynth. Tubes with a total volume of 15 µl were prepared with 3 µl 20 µM Sequencing Primer (1.2 µl 50 µM Primer), and the appropriate amounts of nuclease-free water and plasmid based on the initial concentration of the prepped plasmids. The plasmids were diluted in water to 60 - 80 ng per 15 µl.
Transformations were performed with the method of heat shock. Competent Top 10 cells were, still frozen, inoculated with 10 µl of the desired plasmid, and incubated for 30 minutes on ice. The heat shock was performed for 30 seconds at 42 °C. The cells were incubated for another 2 minutes on ice. 1 ml of SOC medium was added. The cells in the SOC medium were incubated at 37 °C and 300 rpm. Incubation time depended on the used antibiotic. For ampicillin, the incubation time was one hour, for chloramphenicol, kanamycin, and tetracycline the incubation time was two hours. 100 to 300 µl of the cells were spread on an agar plate with the appropriate antibiotic. The plates were incubated overnight at 37 °C.
The protocol of Molecular and Cell Biology, Berkely was followed.
The protocol of Molecular and Cell Biology, Berkely was followed.