Team:Heidelberg/Templates/Methods
From 2013.igem.org
m (→written together: 1st step: Pre-experiment, Reproduction of Paper) |
JuliaS1992 (Talk | contribs) |
||
(7 intermediate revisions not shown) | |||
Line 53: | Line 53: | ||
=== Gibson assembly === | === Gibson assembly === | ||
- | |||
*[[media:Gibson_for_Heidelberg.xls|Excel template]] kindly provided by the [https://2013.igem.org/Team:Freiburg/Project/Coop iGEM Team Freiburg 2013] | *[[media:Gibson_for_Heidelberg.xls|Excel template]] kindly provided by the [https://2013.igem.org/Team:Freiburg/Project/Coop iGEM Team Freiburg 2013] | ||
Line 111: | Line 110: | ||
===Plasmid-DNA isolation=== | ===Plasmid-DNA isolation=== | ||
- | + | ====Miniprep==== | |
+ | <div style="font-size: 14px; | ||
+ | text-align: justify;"> | ||
+ | Plasmids were isolated by the use of the QIAprep® Spin Miniprep Kit. According to the instructions included, all necessary solutions were prepared and steps conducted. First, overnight cultures (1-2 ml) have been pelleted by centrifugation at >8000 rpm</div> | ||
<div style="font-size: 14px; | <div style="font-size: 14px; | ||
text-align: justify;"> | text-align: justify;"> | ||
- | |||
(6800 x g) for 3 min at room temperature (15–25°C). Supernatant was discarded and pellet resuspended in 250 μl (procedure 2: 200 μl) buffer P1 in a microcentrifuge tube. Afterwards 250 μl (procedure 2: 400 μl) Buffer P2 were added and the tube inverted 4-6 times to mix the solution until it became clear. The sample was lysed for 2-3 minutes before adding 350 μl (procedure 2: 300 μl) buffer N3 and inverting the whole solution 4-6 times. Following the neutralization, the sample was centrifuged for 10 minutes at 3,000 rpm (~17,900 x g) in a table-top microcentrifuge. Supernatant was decanted to a QIAprep spin column and vacuum applied to the manifold until the solution was drawn through the column. To wash the column 500 μl Buffer PB were added and again vacuum applied. In addition the QIAprep spin column was washed by adding 750 μl Buffer PE and applying vacuum. To remove residual wash Buffer the column was transfered to a collection tube and centrifuged for 1 min. Finally, the column was put into a clean 1.5 ml microcentrifuge tube and 20-30 μl water (heated up to 55°C) added to the center of the column. After 1 minute the tube containing the column was centrifuged for 1 minute to collect eluted DNA. </div> | (6800 x g) for 3 min at room temperature (15–25°C). Supernatant was discarded and pellet resuspended in 250 μl (procedure 2: 200 μl) buffer P1 in a microcentrifuge tube. Afterwards 250 μl (procedure 2: 400 μl) Buffer P2 were added and the tube inverted 4-6 times to mix the solution until it became clear. The sample was lysed for 2-3 minutes before adding 350 μl (procedure 2: 300 μl) buffer N3 and inverting the whole solution 4-6 times. Following the neutralization, the sample was centrifuged for 10 minutes at 3,000 rpm (~17,900 x g) in a table-top microcentrifuge. Supernatant was decanted to a QIAprep spin column and vacuum applied to the manifold until the solution was drawn through the column. To wash the column 500 μl Buffer PB were added and again vacuum applied. In addition the QIAprep spin column was washed by adding 750 μl Buffer PE and applying vacuum. To remove residual wash Buffer the column was transfered to a collection tube and centrifuged for 1 min. Finally, the column was put into a clean 1.5 ml microcentrifuge tube and 20-30 μl water (heated up to 55°C) added to the center of the column. After 1 minute the tube containing the column was centrifuged for 1 minute to collect eluted DNA. </div> | ||
- | + | ====Midiprep==== | |
<div style="font-size: 14px; | <div style="font-size: 14px; | ||
text-align: justify;"> | text-align: justify;"> | ||
For larger amounts of bacterial cultures the QIAGEN® Plasmid Plus Midi Kit was used to isolate plasmids. According to the instructions included, all necessary solutions were prepared and 25 ml bacterial culture harvested after 12-16 hours incubation. The sample was centrifuged at 6000 x g for 15 min at 4°C and pelleted cells were completely resuspended in 2 ml Buffer P1. Then, 2 ml buffer P2 were added and mixed thoroughly by inverting until the lysate appeared viscous. The solution was incubated at room temperature (15–25°C) for 3 min. The QIAfilter Cartridge was placed into a new tube. Afterwards, 2 ml buffer S3 were added to the lysate and mixed by inverting 4-6 times. The lysate was transfered to the cartridge and incubated at room temperature for 10 minutes. During incubation the QIAGEN Plasmid Plus spin column was put into the vacuum manifold and tube extenders inserted into each column. The plunger was inserted gently into the QIAfilter Cartridge and the lysate filtered into the tube. Thereafter, 2 ml buffer BB were added to the cleared lysate and mixed by inverting 4-6 times. Lysate was then transfered to a QIAGEN Plasmid Plus spin column on the vacuum manifold. Vacuum was applied until all liquid remains were drawn through the column. To wash the DNA 700 μl buffer ETR was added and vacuum applied. Residual wash buffer was removed by centrifugation of the column at 10,000 x g (9,700 rpm) for 1 min in a tabletop microcentrifuge. The column was put into a clean 1.5 ml tube. Finally, 200 μl water (55°C) was added to the center of the spin column. The column was let stand for 1 minute and centrifuged for 1 minute to collect the eluted DNA.</div> | For larger amounts of bacterial cultures the QIAGEN® Plasmid Plus Midi Kit was used to isolate plasmids. According to the instructions included, all necessary solutions were prepared and 25 ml bacterial culture harvested after 12-16 hours incubation. The sample was centrifuged at 6000 x g for 15 min at 4°C and pelleted cells were completely resuspended in 2 ml Buffer P1. Then, 2 ml buffer P2 were added and mixed thoroughly by inverting until the lysate appeared viscous. The solution was incubated at room temperature (15–25°C) for 3 min. The QIAfilter Cartridge was placed into a new tube. Afterwards, 2 ml buffer S3 were added to the lysate and mixed by inverting 4-6 times. The lysate was transfered to the cartridge and incubated at room temperature for 10 minutes. During incubation the QIAGEN Plasmid Plus spin column was put into the vacuum manifold and tube extenders inserted into each column. The plunger was inserted gently into the QIAfilter Cartridge and the lysate filtered into the tube. Thereafter, 2 ml buffer BB were added to the cleared lysate and mixed by inverting 4-6 times. Lysate was then transfered to a QIAGEN Plasmid Plus spin column on the vacuum manifold. Vacuum was applied until all liquid remains were drawn through the column. To wash the DNA 700 μl buffer ETR was added and vacuum applied. Residual wash buffer was removed by centrifugation of the column at 10,000 x g (9,700 rpm) for 1 min in a tabletop microcentrifuge. The column was put into a clean 1.5 ml tube. Finally, 200 μl water (55°C) was added to the center of the spin column. The column was let stand for 1 minute and centrifuged for 1 minute to collect the eluted DNA.</div> | ||
- | + | ====Preparation of large plasmids==== | |
<div style="font-size: 14px; | <div style="font-size: 14px; | ||
text-align: justify;"> | text-align: justify;"> | ||
Line 129: | Line 130: | ||
===Sequencing=== | ===Sequencing=== | ||
Samples were sent to [http://www.gatc-biotech.com/en/index.html GATC Biotech] for sequencing. | Samples were sent to [http://www.gatc-biotech.com/en/index.html GATC Biotech] for sequencing. | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
==Protein detection== | ==Protein detection== | ||
Line 157: | Line 137: | ||
===Purification of Indigoidine and tagged constructs=== | ===Purification of Indigoidine and tagged constructs=== | ||
- | 1 ml of IPTG-induced, blue culture was spun down at full speed (14,000 rpm) for 20 minutes, washed in 1 ml of methanol and centrifuged once more for 5 minutes at 14,000 rpm. Methanol was discarded and samples were dissolved in 200-400 µl DMSO. | + | <div style="font-size: 14px; |
+ | text-align: justify;">1 ml of IPTG-induced, blue culture was spun down at full speed (14,000 rpm) for 20 minutes, washed in 1 ml of methanol and centrifuged once more for 5 minutes at 14,000 rpm. Methanol was discarded and samples were dissolved in 200-400 µl DMSO.</div> | ||
===Purification of Delftibactin=== | ===Purification of Delftibactin=== | ||
Line 185: | Line 166: | ||
====Data Acquisition==== | ====Data Acquisition==== | ||
- | '''Sampling''' | + | |
+ | '''Sampling''' | ||
Two empty 3.2 mm die-cut sheets were prepared from Whatman 903 filter paper. Afterwards, 5 µl of the sample were pipetted on the sheets and dried in the fridge overnight. | Two empty 3.2 mm die-cut sheets were prepared from Whatman 903 filter paper. Afterwards, 5 µl of the sample were pipetted on the sheets and dried in the fridge overnight. | ||
- | '''Sample preparation''' | + | '''Sample preparation''' |
+ | |||
<div style="font-size: 14px; | <div style="font-size: 14px; | ||
text-align: justify;">The dried sheets were extracted for ca. 20 min with 100 μl solution 1. Then the polypropylen microtiter plates were centrifuged for ca. 2 minutes at 4000 rpm. Supernatant was vaporized at 55°C for 30 minutes at the blowing station until all liquid residues were removed. Subsequently the plate was warmed up for 1 minute at a cabinet dryer (65°C). | text-align: justify;">The dried sheets were extracted for ca. 20 min with 100 μl solution 1. Then the polypropylen microtiter plates were centrifuged for ca. 2 minutes at 4000 rpm. Supernatant was vaporized at 55°C for 30 minutes at the blowing station until all liquid residues were removed. Subsequently the plate was warmed up for 1 minute at a cabinet dryer (65°C). | ||
Line 203: | Line 186: | ||
==1st Pre-Experiment== | ==1st Pre-Experiment== | ||
+ | <div style="font-size: 14px; | ||
+ | text-align: justify;">The first step is the establishment of the reaction of delftibactin with soluble gold. Therefore we plan an experiment (one to two weeks), where we reproduce the results of the paper [<bib id="pmid23377039"/>]. After mixing the Acidovorax complex medium (ACM) and adding 4g Chelex-100 resin / 1L ACM we prepare 25 agarplates (0,5 L ) and two media for liquid culture (1L +0,5L). The second day is about the resuspending of the strain D. acidovorans in 5 ml [recepy in methods] and letting it grow over night. The next day 5 agarplates are inoculated with D. Acidovorans by fractional streak and 5 agarplates with streak over half of the plate. In addition to these 10 agarplates, also the two flasks with the liquid media are inoculated with the strain. The rest of the resuspended cells is stored in a glycerol stock. After 3 days of culture growth (shaking at 190rpm, at a temperature of 30°C) the cells are big enough to start with the delftibactin purification (liquid culture) and the fast test on the agarplates. For the fast test on the agarplates we prepare an soft agarose solution (0,5% agarose and 10mM AuCl3) and incubate every plate with 10 ml solution for two hours at rooms temperature. If delftibactin is present there can be observed a ring structure around the cells. </div> | ||
+ | <div style="font-size: 14px; | ||
+ | text-align: justify;">For the purification of delftibactin, the liquid culture will be centrifugated at 7000 rpm for 15 min. The supernatant will be treated with HP20 resin (20 g/L) and then shaken 2h at 220rpm. With the Buchner funnel filtration the resin will be collected and washed with 400 ml dH2O. Afterwards the resin is again washed with 400 ml methanol which will be evaporate under rotary vacuum. The leftover is resuspended in 2 ml of ddH2O:MeOH (1:1). The solution is added to a HPLC column and the HPLC (high performance liquid chromatography) is analysed in a mass spectroscopy facility. The mobile phase consists in 2%-14% acetonitrile and 98% water +5 mM (NH4)2CO3. | ||
+ | After the purification we plan to mix the purified delftibactin with different gold solution (AuCl3) concentrations (between 5mM and 20mM). After a 2h incubation a precipitation would be observed. | ||
+ | In case the pre-experiment works, we have proven the reliability of the paper and the expected read out of the assay.</div> | ||
=== Making ACM Medium === | === Making ACM Medium === | ||
==== for 1 L ==== | ==== for 1 L ==== | ||
Line 229: | Line 218: | ||
=== Reactivation of freeze-dried bacteria === | === Reactivation of freeze-dried bacteria === | ||
* prepare 10 ml of liquid-activation-media with: | * prepare 10 ml of liquid-activation-media with: | ||
- | + | ** 50 mg peptone | |
- | + | ** 30 mg meat extract | |
- | + | ** 150 mg agar | |
- | + | ** 10 ml destilled water | |
- | + | ** adjust pH to 7.0 | |
* rehydrate and grow cells in 5 ml of liquid broth | * rehydrate and grow cells in 5 ml of liquid broth | ||
* grow at 30° in incubator over night | * grow at 30° in incubator over night | ||
Line 257: | Line 246: | ||
* resuspend material in 2 ml 50 % water and methanol | * resuspend material in 2 ml 50 % water and methanol | ||
* liquid chromatography | * liquid chromatography | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
==DNA Storage== | ==DNA Storage== |
Latest revision as of 01:39, 29 October 2013
Cloning
Colony PCR
Colonies were picked with pipet tips and dipped into the PCR mixture.
Ingredient | amount [µl] |
---|---|
Overnight Culture | Top of a pipet tip |
10x PCR buffer | 2.0 |
MgCl2 (25 mM) | 2.0 |
Forward primer (10µM) | 0.2 |
Reverse primer (10µM) | 0.2 |
dNTPs (10 mM) | 1.0 |
Polymerase | 0.5 |
Sterile water | ad 10.0 |
Total | 10.0 |
The following PCR program was adjusted according to annealing conditions.
Cycles | temperature [°C] | Time [min:s] |
---|---|---|
1 | 98 | 2:00 |
35 | 98 | 0:05 |
68 (annealing) | 0:15 | |
72 | 1:00 per kbp | |
1 | 72 | 10:00 |
1 | 12 | inf |
Agarose gel electrophoresis
Gibson assembly
- Excel template kindly provided by the iGEM Team Freiburg 2013
Gibson assembly was prepared according to calculation (see file above). The mixture was incubated at 50°C for 60 minutes and purified by isopropanol precipitation. The Gibson constructs were then transformed into competent bacteria.
Biobrick assembly
- [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf BioBrick_Assembly_Manual.pdf]
Restriction digests
For enzymatic digests, the following formula was used if bovine serum albumin (BSA) was not part of the buffer, which was the case for NEB buffer 3.0:
- ... µl DNA (~500 ng)
- 5 µl BSA
- 5 µl buffer (for example NEBuffer 2)
- 2 x 1.5 µl enzymes (for example EcoRI-HF & PstI)
- ... µl water
=> 50 µl in total
The mixture was incubated at 37°C for 1h or up to 1:30h and analyzed by gel electrophoresis.
In case BSA was already part of the buffer, the following recipe was used:
- ... µl DNA (~500 ng)
- 2 µl buffer (e.g. NEB CutSmart buffer)
- 1 µl restriction enzymes
- water ad 20 µl
The mixture was incubated at 37°C for 1h or up to 1:30h and analyzed by gel electrophoresis.
Extraction of DNA fragments from an agarose gel
Ligation
For ligation of DNA fragments, the mixture was prepared as follows:
- ... µl DNA (=<200ng in total-> 20-70ng Backbone)
- 2 µl buffer
- 1 µl ligase (T4)
- ... µl water
=> 20 µl in total
After an incubation for 2 minutes at room temperature, or for 1h or over night at 4°C, the product was loaded on an gel for electrophoretic separation of the DNA fragments.
Nucleotide Removal
Purification of PCR products
Plasmid-DNA isolation
Miniprep
Midiprep
Preparation of large plasmids
Sequencing
Samples were sent to [http://www.gatc-biotech.com/en/index.html GATC Biotech] for sequencing.
Protein detection
Thin Layer Chromatography
Purification of Indigoidine and tagged constructs
Purification of Delftibactin
- Centrifugation of cultures at 3750 rpm for 30 min
- Retrieve supernatant
- Add 20 g/L HP-20
- Stirr at RT for 2 h
- Filtration with Buchner funnel to retain HP-20
- Wash with 400 ml ddH2O
- Elution with 400 ml Methanol
- Evaporated methanol with rotary evaporator
- Resuspended in 2 ml 50 methanol : 50 ddH2O
Mass spectrometry
Cell preparation
- Pellet was resolubilzed in 500 µl 1x PBS and lysed by ultra-sonification (3x20 seconds; on ice). Finally the sample has been centrifuged and supernatant decanted to 1.5 ml tubes. Frozen with nitrogen the sample was handed over for mass spectrometry.
- Supernatant was frozen with nitrogen and lyophilized for 12 hours ( at ~-45°C and ~0,1 mbar) to remove liquid residues. The lyophilisate was resuspended in 500 microliters methanol and vortexed. Supernatant was transferred to a 1.5 ml tube, which was frozen in liquid nitrogen. The sample was then handed over to mass spectrometry for further analysis.
Data Acquisition
Sampling
Two empty 3.2 mm die-cut sheets were prepared from Whatman 903 filter paper. Afterwards, 5 µl of the sample were pipetted on the sheets and dried in the fridge overnight.
Sample preparation
After drying 60 μl of solution 2 were added and the mircotiter plate tightly closed with micromates. The plate was shaken gently and heated up again for ca. 15 minutes at the cabinet dryer (65°C). Micromates were drawn off and supernatant vaporated on heating plates (55°C surface temperature) with a constant air stream. Again no liquid residues should be remaining. Samples were heated up for 1 minute in the cabinet dryer (65°C).
Finally 100 μl solution 3 were added (it is possible to dilute the sample up to factor 3 with solution 3). To avoid evaporation losses, the microtiter plate was covered with aluminium foil.SDS-PAGE
1st Pre-Experiment
After the purification we plan to mix the purified delftibactin with different gold solution (AuCl3) concentrations (between 5mM and 20mM). After a 2h incubation a precipitation would be observed.
In case the pre-experiment works, we have proven the reliability of the paper and the expected read out of the assay.Making ACM Medium
for 1 L
- 0.5 g yeast extract (Difco-BectonDickinson)
- 1.0 g Cas amino acids (Difco)
- 2.0 g pyruvic acid
- 2.0 g L-glutamine
- 0.3 g KH2PO4
- 0.3 g MgSO4
- 2.0 g MOPS
- 4.0 g Chelex 100-resin (Sigma)
- pH adjusted to 7.2–7.3 with 5 M KOH
Important Notes:
- fill up to 900mL before adding pyruvic acid and L-glutamine
- adjust pH
- fill up to 1L
- treat with Chelex for 1h
- remove Chelex by filtration
Making Plates
- prepare 2 l medium
- make 25 agar plates
- autoclave
Reactivation of freeze-dried bacteria
- prepare 10 ml of liquid-activation-media with:
- 50 mg peptone
- 30 mg meat extract
- 150 mg agar
- 10 ml destilled water
- adjust pH to 7.0
- rehydrate and grow cells in 5 ml of liquid broth
- grow at 30° in incubator over night
Inoculation of agar plates
- inoculate 5 agar plates with inoculation loop
- grow at 30° in incubator for 3 days
Gold-precipitation on agar plates
- prepare 50 ml of 0.5 % Agarose solution which contains 10 mM AuCl3
- pour 10 ml of the prepared agarose solution onto each of 3 agar plates
- recepy for the gold solution media:Recepy_for_0,5%_agarose_solution_with_10mM_AuCl3.pdf
Delftibactin purification
- centrifuge liquid culture at 7000 r.p.m.
- keep supernatant, remove cell mass
- treat supernatant with with 20 g/l HP20
- shake HP20 with supernatant for 1 h at 220 r.p.m.
- remove aqueous phase by buchner vacuum filtration
- elute dehydrated material with 400 ml methanol
- remove HP20 by buchner vacuum filtration
- dry solution with rotary vacuum evaporator
- resuspend material in 2 ml 50 % water and methanol
- liquid chromatography
DNA Storage
Plasmid Shipping
- uses Whatman Filter Paper
- used the protocol of: https://labs.mcdb.ucsb.edu/weimbs/thomas/links/methods/plasmid_shipping.html
Sending
- Mark a circled area with a pencil (not a marker pen) on a clean Whatman #1 filter paper (or equivalent).
- Spot about 2 µg of plasmid DNA into the circle. Allow the filter paper dry at room temperature.
- Insert spotted filter paper inside a plastic bag and heat-seal it.
- Send by regular (air) mail.
Receiving
- To recover the DNA, use clean gloves and cut the marked circle area that contains the dried plasmid DNA.
- Using clean forceps, insert the filter paper into a 1.5 ml micro centrifuge tube. Add 100 µl of TE buffer, vortex briefly and incubate at room temperature for 5 minutes. Vortex again and centrifuge the tube for a few seconds.
- Remove about 10 µl of supernatant for use in transfecting E. coli by electroporation or chemical means. Please do not try to use the DNA directly for any application other than to transform bacteria and prepare a plasmid stock.
- Store the remainder of the filter paper/TE mix at -20 or -80 C as a permanent archive in case that your plasmid stock ever gets lost or if something turns out to be wrong with it.
Electrocompetent Bacteria
Materials
- 10% glycerol
- ddH2O
Day 1
- Inocculate 50 ml of appropriate medium
- Grow ON at appropriate temperature, shaking (e.g. 37°C, 180 rpm)
Day 2
- Transfer 10 - 30 ml of the ON culture into 400 ml appropriate medium (measure OD600, should not exceed 0.1)
- Grow at appropriate temperature, shaking (e.g. 37°C, 180 rpm) until an OD of 0.4-0.6 is reached
- Meanwhile, put H2O, 10% glycerol on ice, prechill tubes and centrifuge
- Chill cells on ice for 10 min
- Centrifuge for 30 min at 3750 rpm, 4°C
work on ice from here on - Discard supernatant, resuspend bacteria in 2 ml ice cold H2O / 50 ml falcon, fill up to 20 ml ice cold H2O
- Centrifuge for 30 min at 3750 rpm, 4°C
- Discard supernatant, resuspend in 1 ml glycerol / falcon, add up to 5 ml glycerol (falcons may be pooled at this point)
- Centrifuge for 20 min at 3750 rpm, 4°C
- Resuspend in same volume glycerol as pellet size (ca. 100 µl / original 50 ml falcon)
- Aliquot 50 µl per tube
- Optional: shock freeze with liquid nitrogen
- Store aliquots at -80°C
Electroporation of E.coli DH10β
Preparation:
- Source material: 1ml LB media + DH10β overnight culture (12-16 h) at 32°C (1 day before)
- Preperate 4 eppis with 1 ml LB media and a) 25µl, b) 35µł, c) 45µł, d) 55µl of source material and incubate it at 32°C (2h)
- Put bacteria on ice for 10 – 15 min (since this step everything is at 0°C – the centrifuge, the water!!!!!!!!)
- Centrifuge 30 s at 13 000 rpm (0°C) and remove via vacuum the medium, DON'T touch the pellet!!!
- Resuspend first pellet in 1 ml H2O bidest comercial (0°C),transfer to next eppi an dos on to collect all four cell-pellets resuspended in water in 'ONE eppi.
- Centrifuge and wash again (2x)
Procedure
- add 50 µl H2O bidest comercial (0°C) and resuspend pellet
- add 2-3 µl of Ligation Reaction and incubate it on ice for 5-10 min
- transfer all of it to the special cuvette (without any air bubbles) – the cuvette has to be perfectly dry on the outside
- press button – electroshock (and from now on QUICK handling)
- add 400 µl SOC-medium and transfer all of it into an eppi
- incubation for 1 h at 37 °C shaking at 240 rpm
plate
- plate on one agarplate with the corresponding antibiotic 100 µl of the bacteria
- plate on another agarplate with the corresponding antibiotic 10 µl of the bacteria
Additionally
- Bio-Rad MicroPulser
- Program 1 for brown cuvettes
- Program 2 for blue cuvettes
=> these two programs are for bacteria
Heat shock competent bacteria
Materials
- 100 mM CaCl2 (aq)
- Glycerol
Day 1
grow bacteria over night in 50 ml medium at 37°C
Day 2
- transfer 20-30 ml of the ON culture into 400 ml LB
- grow at 37°C until an OD of 0.5-0.6 is reached (3-5 hours), put CaCl2 on ice, prepare aliquoting buffer (20 ml CaCl2 with 10% glycerol)
- centrifuge bacteria for 30 min at 3750 rpm, 4°C
work on ice from here on
==> you will need 8 falcons (50 ml) in order to do so - discard supernatant, resuspend bacteria in 5 ml CaCl2 / falcon, pool 2 falcons, add up to 50 ml CaCl2
==> you should have 4 falcons from here on - leave on ice for 30 min
- centrifuge for 20 min at 3750 rpm, 4°C
- discard supernatant, resuspend in 10 ml aliquoting buffer for all 4 falcons, add another 10 ml aliquoting buffer
Do not vortex - pipet 100 µl per eppendorf tube (pre-cooled on ice), put aliquots in -80°C freezer
Isopropanol PCR Purification
- pool PCR reactions if there are several
- add >= 4 volumes isopropanol, invert tube 10 times
- centrifuge 20 min at full speed (use cooling centrifuge at 20°C)
- decant supernatant
- add 4 volumes 70% ethanol
- centrifuge 5 min at full speed
- decant supernatant, tap eppendorf tube on paper tissue
- let it dry for ca. 5 min
- dry off inside of tube with a piece of paper tissue. Do not touch pellet!
- resuspend in 130 µl H2O, add 15 µl CutSmart buffer and 5 µl DpnI (digests methylated DNA -> template)
- incubate 4h at 37°C
- repeat purification, resuspend in 30-40 µl H2O
PCR
- ... µl DNA (1-10ng)
- 2x 0,5 µl Primer
- 25 µl Phusion Flash Ready Mix(2x)
- ... µl water
=> 50 µl in total
- fill in PCR tubes
- PCR amplification takes ca. 2h
validation PCR
( using 2x Taq MasterMix from NEB )
- ~ 5 ng template DNA (picked colony, miniPrep...)
- 2x 0.2 µl primer (_fw, _rv)
- 10 µl 2x Taq MM
- ... H_2O (add up to 20 µl)
=> 20 µl in total
- fill in PCR tubes
- PCR amplification takes ca. 2h
Preparing Glycerol Stock
- grow liquid culture of the desired bacteria
- pipet 1 ml of liquid culture into cryotube, add 300 µl 50% glycerol
- RT for 1 h
- freeze at -80°C
Software
Main packages
- Python(2.7): The high-level programming language [http://www.python.org/ python] was used in combination with Django to develop the web-based interface and client-side applications.
Packages, plugins or libraries loaded by some of our python functions
- Requests: The purpose of this library is to do simple http requests.
- Biopython (>= 1.61): Just like bioperl this package contains tools for computational molecular biology.
- xhtml2pdf: The converter package xhtml2pdf was applied to generate pdf from HTML/CSS templates.
- BeautifulSoup: The BeautifulSoup HTML/XML parser was used for quick-turnaround applications.
- celery: Asynchronous scheduling framework.
- kombu: Database-based message passing framework.
- python-openbabel(>= 2.3.2) : Python bindings to openbabel for 2D (chemical) structure generation.
- UNAFold and MFold utils: UNAFold was required by Gibthon for DNA/RNA folding predictions.
- MySQL: The relational database management system [http://www.mysql.com/ MySQL] was utilized for database creation and maintenance.
- MySQL - Python connector: A conncetor between the programming language python and the database management system.
- Django(>= 1.5): The web application framework Django was used to achieve an easier database connection and is based on python. Since we improved and added basic functions of Gibthon (iGEM Team Cambridge 2010), the integration was easier to handle using the same frame work.
Django packages
- django-registration: Handles user registrations and logins.
- django-annoying: Gibthon uses this package. It is thought to eliminate annoying things in Django.
- south: The south package handles database migration (database independent).
- django-celery: The package was used for the integration of Celery (asynchronous task queue/job queue) into Django.
C++
- CMake: Cross platform build system used by the NRPSDesigner.
- MySQL C++ connector: C++ library for MySQL access.
- libXML: C library for XML handling.
- libcurl: C library for client-side URL transfers.
- Boost.program_options: C++ library for command-line option parsing.
- GCC (>= 4.8) or a comparable compiler with C++11 support.
Javascript
- Intro.js: This Javascript has been adopted for the guided step-by-step tour on the NRPS Designer homepage.
- select2.js: The Select2 library is a jQuery based library for the replacements of select boxes, which support different connections to other functions.