NB: because of the lack of a right 42°C water bath, the heatshock step was made at 37°C for 40 sec.
2 different plates were made: one at [Cm] = 25 g/L and the other at [Cm] = 12.5 g/L
The gel is not that clear: pSB1C3 without the insert is to high on the gel, but our transformant is a the good weight length : 3k3 bp. We will need to investigate further in order to be sure that we really have pSB1C3 + VhCqsA.
6 Clones, 2 from CM25 (25-1 and 25-2) and 4 from Cm 12.5 (12-1, 12-2, 12-3 and 12-4 ) that appear on Cm25 and Cm12.5 were grow on plate and on liquid media (5mL) LB + Cm 25 in order to miniprep them with the Miniprep protocol
Digestions were made on the plasmids obtained with the digestion protocol.
We identified a insert on our native pSB1C3, of around 600 bp, matching with the previous gels that we had. Also, 3 clone are ok: Clone1, 25-1 and 25-2. All of them have the wanted digestion: 2000 bp + 1300 bp. We will further investigate using PvuII, cutting twice, once on the insert, once on the vector.
In order to confirm the insert, two more digestion were made, with PvuII (which cut once on pSB1C3, and not on the insert) and SacI (which cut once on the insert, and once on pSB1C3) following the digestion protocol
The 3 plasmids were cut three time, as it was expected for the one containing CqsA sequence. Those 3 clone are validated, and have been stored on glycerol 25%.
Their name will become: CqsA Anna (25-1), CqsA Maxant (C1) and CqsA Teo (25-2 )
Cloning of Vc_CqsA
The gel extraction from the 08/10 was digested using EcoRI and SpeI with the digestion protocol. The quantities for QSP 100 were:
2.5µL of EcoRI
2.5µL of SpeI
30µL of DNA template
10µL of Cutsmart
55µL of water mQ
No heat inactivation were made. Instead, a PCR puriffication was processed using Sigma GenEtutTM PCR Clean Up kit. Afterward, a DNA quantification gel were made containing:
2µL of ladder 1kb
1µL of loading dye + 5µL of digested DNA
1µL of loading dye + 5µL of digested pSB1C3
First, it is clear that the gel extraction worked fine. Second, it seems that the insert is twice the concentration of the digested plasmid.
Consequently, the ligation was carried out and lasted 50min. The followig quantities were used :
100µL of competent cells + 5 µLof TE buffer + 5µL of DNA: plate B
100µL of competent cells + 9µL of TE buffer + 1µL of pSB1C3 non digested (positive control): plate C
After O/N incubation, the followging observation were made on the Petri dishes:
Plate A: 12 transformants
Plate B: 19 transformants
Plate C: cell mat (even with a dilution x10)
Hence, eight transformants from each plate were put on liquid LB-Cm medium for subsequent mini prep. They were named A 1 to 8 and B 1 to 8. The transformants named were minipreped. The resulting DNA solutions were stored at -20°C.
After a quick analysis gel of all the miniprep, without any digestion (data not shown), 6 transformants seems to have the right plasmid lenght: A3, A4, A5, B3, B5, B6.
Thus, these transformants were digested with PvuII and E/P to assess the right insertion of Vc CqsA.
Premix for PvuII (for 8 digestions):
2 µL of pSB1C3 E/S + 8 µL of PvuII premix
2 µL of A3 miniprep + 8 µL of PvuII premix
2 µL of A4 miniprep + 8 µL of PvuII premix
2 µL of A5 miniprep + 8 µL of PvuII premix
2 µL of B3 miniprep + 8 µL of PvuII premix
2 µL of B5 miniprep + 8 µL of PvuII premix
2 µL of B6 miniprep + 8 µL of PvuII premix
Each tube was put in a water bath at 37°C for 1h. The resulting digestion were put on a 1% agarose gel as follows:
All of the transformants have the same profil and have both of the expected bands. Finally, the 6 transformants are 6 right clones Vc CqsA.
Clones A4 and A5 were named Vc CqsA 1 and Vc CqsA 2 and put in liquid culture to be stored at -80°C the next day.
Solid Bioluminescence assay
Four E. coli precultures were made in LB-Cm (5 mL):
Vh CqsA Max MG1655
Vh CqsA Anna MG1655
Vh CqsA Teo MG1655
pSB1C3 empty MG1655
Each tubes were complemented with 208 µL of a sterile glucose solution to reach a final concentration of 10 g/L. The tubes were put at 37°C O/N. In addition, two V. harveyi precultures were made:
LB: Vh WT (5 mL)
LB: JMH626 (10 mL)
They were put at 30°C O/N. OD of the O/N precultures were taken at 8.06 am. Therefore, fresh LB-Cm-Glc flasks of 10 mL were inoculated to reach OD = 0.1. (final glucose concentration in the medium: 10 g/L). Time of inoculation: 8.32 am. As OD = 0.3 had already been passed at 10.40 am, IPTG induction was made immediatly (final concentration : 0.5 mM). At 1 pm, the cultures were at the end of their exponential phase. Thus, the supernatants could be retrieved following the next steps:
pipeting of 10 mL of each of the cultures in (x5) eppendorf tube of 2 mL
centrifugation max speed, 5 min
pooling of each resulting supernatants
filtration through a 0.2 µm filter
storage at -20°C
OD of the V. harveyi O/N precultures were checked again at 11.10 am: JMH626: 4.15, Vh WT:3.11
(x5) LB flasks of 10 mL were inoculated with JMH626 to reach OD = 0.1. In addition, (x1) LB flask of 10 mL was inoculated with Vh WT, also at OD = 0.1. Time of inoculation: 11.28 am. When the OD was around 0.7, each of the 10 mL cultures were centrifugated at 4500 rpm for 6 min. The resulting supernatants were discarded while the pellets were resuspended with 5 mL of fresh LB medium and 5 mL of SN (one flask = one SN). Then, the cultures were put at 30°C. The whole process of resuspension was over at 3.30 pm.
In addition, 80 µL of the resuspended JMH cultures were dropped-off on a Petri dish and put at 30°C O/N. Acting as a landmark of bioluminescence, 80 µL of a Vh WT culture were also put on the Petri dish. For liquid cultures, the positive control showed bright bioluminescence as expected. Meawhile, all the cultures with the clones SN stayed dark. Regular checkings didn't bring more information. The Petri dish incubated O/N at 30°C was observed.
Test plan of the Petri dish (The Vh WT drop is the bioluminescence landmark)
The positive control, JMH626 culture supplemented with Vh WT SN is as bright as the Vh WT drop. Thus, SN from Vh WT can restore bioluminesence to its normal state, as far as human eyes can tell. The negative control shows faint bioluminscence, corresponding either to a basal expression of quorum sensing, or an activation of the quorum sensing by the HA1 ad AI-2 molecules produced naturally by E. coli. The second option seems quite unlikeky as LuxN and LuxQ (receptors of HA1 and AI-2 respectively) are knocked out in the JMH626 genome. SN of clones A and T show a bioluminescence as strong as the negative control, no real activity of C8-CAI-1 can be observed. SN of clone M seems to restore bioluminescence to normal state, as for the positive control.
Additional experiments need to be performed to conclud on these bioluminescence essays. Particularly, bioluminescence of JMH626 without any supplementation has to be tested.
Digestion, ligation and transformation of Vh1-pBR322
Analytical gel: digestion control (07/17)
06/20: pBR322 (amplified the 06/19) was digested with EcoRI-HF and PstI-HF. Gel migration and gel extraction were performed to keep the vector.
07/17: Vh1 part (PCR from IDT part) was digested with EcoRI-HF and PstI-HF, and purified with PCR purif kit.
07/20: Ligation with T4 DNA Ligase and associated buffer from New England Biolabs was performed with pBR322 and Vh1 both digested with EcoRI-HF and PstI-HF, and the ligation mix was transformed into E. coli Stellar competent cells.
07/21: 6 transformants were observed.
07/24: The 6 transformants were grown on liquid culture for plasmid extraction.
07/25:Plasmids were extracted with miniprep kit. Analytical digestion was performed with EcoRI/PstI and PvuII/XhoI. Three transformants had the expected digestion profile and were stored for Vh1-Vh2-pBR322 cloning.
Digestion, ligation and transformation of Vh2-pBR322
Analytical gel before ligation (07/18)
06/20: pBR322 (amplified the 06/19) was digested with EcoRI-HF and PstI-HF. Gel migration and gel extraction were performed to keep the vector.
17/07: Vh2 part (PCR from IDT part) was digested with EcoRI-HF and PstI-HF, and purified with PCR purif kit.
18/07: Ligation with T4 DNA Ligase and associated buffer from New England Biolabs was performed with pBR322 and Vh1 both digested with EcoRI-HF and PstI-HF, and the ligation mix was transformed into E. coli Stellar competent cells.
07/26: Observed transformants were grown on liquid culture.
07/26: Plasmids were extracted with miniprep kit. Analytical digestion was performed with EcoRI/PstI and PvuII/XhoI. All the transformants had the expected digestion profile and were stored for Vh1-Vh2-pBR322 cloning.
Digestion, ligation and transformation of Vh3-pSB1C3
Analytical gel before ligation (07/12)
08/12: Vh3 part (PCR from IDT), previously digested EcoRI-HF and PstI-HF, was digested with EcoRI-HF and SpeI-HF and purified with PCR purif kit. pSB1C3-Ter (BBa_1006) was digested with EcoRI-HF and SpeI-HF and gel extracted. Ligation with T4 DNA Ligase and associated buffer from New England Biolabs and transformation were performed. E. coli Top10 competent cells previously prepared were used.
08/14: Eight transformants were observed on the transformation plates and six of them were grown on liquid culture.
08/15: Plasmid extraction with miniprep kit and analytical digestion of the 6 transformants with EcoRI/PstI and ApaI/NcoI. The two transformants with the expected digestion profile were stored for cloning into conjugative plasmid and for diacetyl production in E. coli.
Digestion, ligation and transformation of Vh1-Vh2-pBR322
08/07: Vh1-pBR322 and Vh2-pBR322 were digested with EcoRI-HF/XhoI (EX) to try the ligation Vh1 part into Vh2-pBR322. Gel migration and gel extraction were performed with these digestion mix.
08/17: Ligations was performed: Vh1 EX (insert) with Vh2-pBR322 EX (vector). T4 DNA Ligase and associated buffer from New England Biolabs were used. E. coli Top10 competent cells previously prepared were used for transformation of ligation mix.
08/18: Transformants were observed on the transformation plate. They were grown on liquid culture for plasmid extraction and analytical digestion.
08/19: Plasmid extraction was performed with miniprep kit for all the transformants. The plasmids were then digested with EcoRI-HF/PstI-HF and BamHI/XhoI. All the transformants had the expected profile. They were stored for a Vh1-Vh2-Vh3 cloning.
Digestion, ligation and transformation of RFP-pBBR1MCS-4 and RFP-pBBR1MCS-5
Analytical gel before ligation (13/09)
pBBR1MCS-4 (1) and pBBR1MCS-5 (1) were used for ligation
08/15: Plasmid extraction was performed using miniprep kit. Preparative digestion of the conjugative plasmids pBBR1MCS-4 and pBBR1MCS-5 with EcoRI-HF and SpeI-HF (ES) from New England Biolabs was performed.
09/14: Ligation with T4 DNA ligase and associated buffer from New England Biolabs was performed with pBBR1MCS-4 ES and pBBR1MCS-5 ES (vector) with RFP ES (BBa_J04450, insert). The RFP was initially digested by the Pichia pastoris module for its own constructions. The ligation mix was transformed into E. coli Top10 competent cells previously prepared.
09/19: Red transformants were observed on the transformation plates for ligation RFP + pBBR1MCS-4 and RFP + pBBR1MCS-5. They were grown on liquid culture, and the two strains (one for pBBR1MCS-4 and one for pBBR1MCS-5) which seem to have the higher RFP activity were used for conjugation.
Conjugation of RFP-pBBR1MCS-4 and RFP-pBBR1MCS-5 in Vibrio harveyi JMH626
09/21: E. coli pBBR1MCS-4 AmpR, E. coli pBBR1MCS-5 GmR, V. harveyi JMH626 CmR KanR and E. coli pRK2073 SpcR helper were grown overnight on 5 mL liquid culture with appropriate antibiotics. E. coli were grown at 37°C and V. harveyi at 30°C.
09/22: Centrifugation and resuspension in LB was performed for each liquid culture. The following conjugation mix were prepared:
Conjugation AmpR: E. coli pBBR1MCS-4 - RFP + V. harveyi JMH626 + E. coli pRK2073
Conjugation GmR: E. coli pBBR1MCS-5 - RFP + V. harveyi JMH626 + E. coli pRK2073
Negative control AmpR: E. coli pBBR1MCS-4 - RFP + E. coli pRK2073
Negative control GmR: E. coli pBBR1MCS-4 - RFP + E. coli pRK2073
Each mix was deposed on a membrane upon a LB plate. The plates were incubated overnight at 30°C.
09/23: The membranes were resuspended into water and the suspensions were spread on a new LB plate with double antibiotic selection: Amp and Cmp for “Conjugation AmpR” and “Negative control AmpR”, Gm and Cmp for “Conjugation GmR” and “Negative control GmR”. The plates were incubated overnight at 30°C.
09/24: An uncountable quantity of colonies was observed on the two conjugation plates. No colonies were observed on the negative controls, which proves that conjugation worked. The cells were not red yet, and the contact inhibition on the plate (because of the too high quantity of colonies) may alter it aspects. Thus, the conjugation cells were replated on another plate with the same double antibiotic selection.
09/25: The replated Vibrio harveyi cells showed a red fluorescent activity compared to a plate with V. harveyi JMH626 without conjugation.
Plates obtained from conjugation Left: negative control / center: conjugation / right: replated conjugation colonies Above: with pBBR1MCS-4 / Below: with pBBR1MCS-5
Comparison of JMH626 from conjugation (RFP-pBBR1MCS-4) and JMH626 (no conjugation)
Comparison of JMH626 from conjugation (RFP-pBBR1MCS-5) and JMH626 (no conjugation)
09/26: V. harveyi JMH626 with pBBR1MCS-4, V. harveyi JMH626 with pBBR1MCS-5, V. harveyi JMH626 without plasmid (control) were grown on liquid culture overnight with antibiotics and LB at 30°C.
PCRs were processed on the pPICZαA coming from miniprep (cf section 1): PCR protocol
The aim of this PCR was to obtain a linear plasmid with 2 specific restriction sites at its extremity (BamHI and KpnI).
Once the PCR was over a gel migration (at 100 V, during 30 min) was performed: Gel migration protocol
pPICZα was not pure but we decided to do a gel extraction of the band of interest after the digestion. So PCR products went through a PCR purification.
Nanodrop of the PCR product: [pPICZαA] = 260,5 ng/µL
Once we knew the DNA concentration we decided to do the preparative digestion (BamHI-KpnI-HF): Digestion protocol
A gel migration (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol
PCRs was processed on the part pGAP-Odr10-pFUS1-cOT2 received from IDT: PCR protocol
Once the PCR was over a gel migration (at 100 V, during 30 min) was performed: Gel migration protocol
Odr10-cOT2 was not pure but we decided to do a gel extraction of the band of interest after the digestion. So PCR products went through a PCR purification.
Nanodrop of the PCR product: [Odr10-cOT2] = 277,3 ng/µL
Once we knew the DNA concentration we decided to do the preparative digestion (BamHI and KpnI-HF): Digestion protocol
A gel migration (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol
Finally, competent cells transformation of E. coli DH5α was processed following the protocol: Transformation protocol. (NB: competent cells were plated on LB medium [zeo] = 25 g/L.)
Several clones were put in liquid culture of 5 ml of LB with [zeo] = 25 g/l.
Restriction map of transformants
Plasmids extraction was performed on the previous culture of 5 mL E. coli transformants grown on LB liquid media with zeocin: Miniprep protocol
The 4 400 pb band of Odr10-cOT2 was purified: pPICZα-pFUS1
The 400 pb band of Odr10-cOT2 was purified: cOT2
The 2 400 pb band of pGAP-leucro was purified: pPICZα-pGAP
The 300 pb band of pGAP-leucro was purified: leucro
The 300 pb band of pGAP-DNY15 was purified: DNY15
A gel migration (at 100 V, during 30 min) was performed, in order to quantify plasmids and inserts: Gel migration protocol
Ligation & transformation of DNY15; cOT2 and leucrocine in pSB1C3 (Ligation Protocol with T4 DNA Ligase (M0202))
Finally, competent cells transformation in E. coli DH5α was processed following the protocol: Transformation protocol. NB: competent cells were plated on LB medium [cm] = 25 g/L. 6 clones of each plate were grown into LB+Chloramphenicol (5 ml each tube).
Plasmids extraction was performed on the previous culture of 5 mL E. coli transformants grown on LB liquid media with chloramphenicol.
A gel migration (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol. All clones with good gel migration profile were kept.
Integration of genic construction in P. pastoris genome
To integrate a genic construction in P. pastoris genome, the first step is to linearize the plasmid at the localization of the integration (for instance in our case we linearize in the GAP promotor to have an integration in this promotor).
pPICZα without insert was used as a negative control.
Once digested, each construction is electropored in P. pastoris following the Electroporation protocol.
Yeast after transformation were plated on YPD + [zeo] = 250 g/l. Every transformants were then replated on YPD + [zeo] = 1000 g/l to select clones with a higher rate of genomic integration.
pGAP-DNY15: A gel migration to amplify the pGAP-DNY15 gene integrated in pichia genome (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol
Odr10-cOT2: A gel migration to amplify the Odr1 0-cOT2 gene integrated in pichia genome (at 100 V, during 30 min) was performed, in order to separate the DNA fragments: Gel migration protocol
RT-PCR experiment
The P. pastoris strains containing pGAP-DNY15 or the empty plasmid were grown in 50mL YPD 40g/L of glucose for 4 days at 30°C in an agitating incubator. RNAs of both were extracted and a RT-PCR experiment has been done.
AMP production and cytotoxicity tests
D-NY15 production was performed with the P. pastoris clone E obtained previously. Two cultures were carried out: one for D-NY15 E and the other for the negative control (P. pastoris transformed with pPICZα without insert). Each clone was inoculated in 50 mL YPD 40 g/L glucose and grown for 4 days at 30 °C in an agitating incubator. 15mL of each supernatant culture were stored at 4°C while 35mL were freeze-dried and then resuspended in 3.5mL of water.
Cytotoxicity test on plate were made using the disc diffusion technique. 200µL of V. harveyi WT (OD600 around 0.5) 100 times diluted were spread on LB agar. Discs were soaked with supernatant, placed on the petri dish and incubated overnight at 30°C.
The same experiment was made again spreading 200µL of V. harveyi WT no diluted:
Protocols
PCR
Introduction
We used the Thermo Scientific Phusion High-Fidelity DNA Polymerase. Amplification of templates with high GC content, high secondary structure, low template concentrations or long amplicons may require further optimization.
Materials
PCR thermocycler
PCR tubes
nuclease-free water
dNTP
Phusion HF Buffer (X5) or GC Buffer
Primers (both forward and reverse)
Template DNA
Phusion polymerase
Procedure
All components should be mixed and centrifuged prior to use. It is important to add Phusion DNA Polymerase last in order to prevent any primer degradation caused by the 3´→ 5´ exonuclease activity.
Phusion DNA Polymerase may be diluted in 1X HF or GC Buffer just prior to use in order to reduce pipetting errors.
Use of high quality, purified DNA templates greatly enhances the success of PCR.
We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C).
Component
50 μL
final concentration
Nuclease-free water
qs 50 μL
Buffer Phusion HF (5X)
10μL
1X
10 mM dNTPs
1 μL
200 μM
10 μM Forward primer
2.5 μL
0.5 μM
10 μM Reverse Primer
2.5 μL
0.5 μM
DNA template (10 ng/μL)
1 μL
10ng
Phusion DNA Polymerase
0.5 μL
1.0 U/0.5 μL of reaction
Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary
Transfer PCR tubes from ice to a PCR machine with the block preheated to 98°C and begin thermocycling:
Then purify the products thanks to PCR purification kit
PCR purification
Introduction
This protocol was extracted from Invitrogen PureLink® PCR Purification Kit. Refer to this protocol for troubleshooting. Use the PureLink® PCR Purification Kit to efficiently remove primers, dNTPs, enzymes, and salts from PCR products in less than 15 minutes. Use the kit with Binding Buffer High-Cutoff (B3) to remove primer dimers or short spurious PCR products. The purified PCR product is suitable for automated fluorescent DNA sequencing, restriction enzyme digestion, and cloning.
Materials
Binding Buffer (B2)
Binding Buffer High-Cutoff (B3)
Wash Buffer (W1)
Elution Buffer; 10 mM Tris-HCl, pH 8.5 (E1)
PureLink® PCR Spin Columns with Collection Tubes
PureLink® Elution Tubes (1.7 mL)
50–100 μL PCR product
100% isopropanol
96–100% ethanol
Sterile, distilled water (pH>7.0)
Microcentrifuge capable of achieving >10,000 × g
Procedure
/!\ The PureLink® PCR Purification Kit buffers contain guanidine hydrochloride and isopropanol. Always wear a laboratory coat, disposable gloves, and eye protection when handling buffers.
/!\ Do not add bleach or acidic solutions directly to solutions containing guanidine hydrochloride or sample preparation waste because it forms reactive compounds and toxic gases when mixed with bleach or acids.
Follow the recommendations below to obtain the best results:
Maintain a PCR volume of 50–100 μL
Save an aliquot of PCR products before purification to verify and check the amplicon on the gel
Use a centrifuge at room temperature for all steps
Pipet the Elution Buffer (E1) in the center of the column and perform a 1 minute incubation
Always use sterile water with pH 7–8.5, if you are using water for elution
Before starting. Add isopropanol to the Binding Buffers and ethanol to the Wash Buffer according to the following table. After adding isopropanol or ethanol, store all buffers at room temperature.
Buffer
Cat. no. K3100-01
Binding Buffer (B2)
10mL 100% isopropranol
Binding Buffer HC (B3)
2.3mL 100% isopropranol
Wash Bufer (W1)
64mL 96-100% isopropranol
Binding DNA.
Add 4 volumes of PureLink® Binding Buffer (B2) with isopropanol (see before starting) or Binding Buffer HC (B3) with isopropanol (see before starting) to 1 volume of the PCR product (50–100 μL). Mix well.
Remove a PureLink® Spin Column in a Collection Tube from the package.
Add the sample with the appropriate Binding Buffer (from step 1 of this procedure) to the PureLink® Spin Column.
Centrifuge the column at room temperature at 10,000 × g for 1 minute.
Discard the flow through and place the spin column into the collection tube.
Washing DNA
Add 650 μL of Wash Buffer with ethanol (see before starting) to the column.
Centrifuge the column at room temperature at 10,000 × g for 1 minute. Discard the flow through from the collection tube and place the column into the tube.
Centrifuge the column at maximum speed at room temperature for 2–3 minutes to remove any residual Wash Buffer. Discard the collection tube. Then let the residual ethanol evaporate by placing the open column on the collection tube and let it sit for 5 mins.
Eluting DNA.
Place the spin column in a clean 1.7-mL PureLink® Elution Tube supplied with the kit.
Add 30 μL of Elution Buffer (10 mM Tris-HCl, pH 8.5) or sterile, distilled water (pH >7.0) to the center of the column.
Incubate the column at room temperature for 1 minute.
Centrifuge the column at maximum speed for 2 minutes.
The elution tube contains the purified PCR product. Remove and discard the column. The recovered elution volume is ~48 μL. Store the purified PCR product at –20°C or use the PCR product for the desired downstream application.
Colony PCR
Introduction
This protocol was elaborated thanks to the help of Anthony Henras.
Materials
10 μL of 0.02N NaOH / 1 PCR
Procedure
Resuspend the equivalent of the tip of a P1000 pipette of the colony in 10 μL of 0.02N NaOH
Mix well (vortex)
Incubate 5 min at 95°C and then chill on ice for 10 min at 4°C (program the thermocycler to do so (Program YeastLysis))
For each PCR mix: NOTE: mix on ice and put on the thermocycler directly after mixing
Component
Volume (μL)
Previous cell extract
2
Taq Pol Buffer
10
Forward oligo 100 10 μM
0.5
Reverse oligo 100 10 μM
0.5
dNTP
1
H2O
35.6
Taq DNA polymerase
0.4
Put on a thermocycler and start this cycle:
95°C
5 min
35 cycles
95°C
30 sec
55°C
1 min
72°C
3 min
72°C
10 min
22°C
∞
Migration on gel to check the results
Gel extraction
Procedure
Please, before doing your preparative gel, use one sample to make an analityc one !
Equilibrate a water bath or heat block to 50°C.
Excise a minimal area of gel containing the DNA fragment of interest.
Crucial: To protect the UV box, it is a good idea to place the gel on a glass plate if available.
Try to get as little excess gel around the band as possible.
Weigh the gel slice containing the DNA fragment using a scale sensitive to 0.001 g.
Add Gel Solubilization Buffer (L3) to the excised gel in the tube size indicated in the following table:
Gel
Tube
Buffer L3 Volume
≤2% agarose
1.7 mL polypropylene
3:1 (i.e., 1.2 mL Buffer L3: 400 mg gel piece)
>2% agarose
5 mL polypropylene
6:1 (i.e., 2.4 mL Buffer L3: 400 mg gel piece)
Place the tube with the gel slice and Buffer L3 into a 50°C water bath or heat block. Incubate the tube at 50°C for 10 minutes. Invert the tube every 3 minutes to mix and ensure gel dissolution.
Note: High concentration gels (>2% agarose) or large gel slices may take longer than 10 minutes to dissolve.
After the gel slice appears dissolved, incubate the tube for an additional 5 minutes.
Optional: For optimal DNA yields, add 1 gel volume of isopropanol to the dissolved gel slice. Mix well.
Before Starting: Add ethanol to the Wash Buffer (W1) according to the label on the bottle.
Purifying DNA Using a Centrifuge
Load. Pipet the dissolved gel piece onto a Quick Gel Extraction Column inside a Wash Tube. Use 1 column per 400 mg of agarose gel.
Note: The column reservoir capacity is 850 μL.
Bind. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube.
Wash. Add 500 μL Wash Buffer (W1) containing ethanol to the column.
Remove Buffer. Centrifuge the column at >12,000 × g for 1 minute. Discard the flow-through and place the column into the Wash Tube.
Remove Ethanol. Centrifuge the column at maximum speed for 1–2 minutes. Discard the flow-through.
Elute. Place the column into a Recovery Tube. Add 30 μL Elution Buffer (E5) to the center of the column. Incubate the tube for 1 minute at room temperature.
Collect. Centrifuge the tube at >12,000 × g for 1 minute.
Store. The elution tube contains the purified DNA. Store the purified DNA at 4°C for immediate use or at −20°C for long-term storage.
Gel migration
Introduction
This protocol is the classical one used for electrophoresis. - You can adapt the concentration of agar according to the length of your fragment 1% agar if the DNA fragments are big 2% agar if the DNA fragments are small (the bigger fragment are sticked together) - Adapt the volume of the gel 15 to 30 mL for small gels and 150 to 200 mL for big gels
Procedure
Thoroughly rinse gel housing and well-comb with dH2O.
Place gel mold perpendicular to flow direction, ensuring proper sealing of rubber gaskets.
Add the calculated amounts of 0.5xTBE and agarose to a fresh Erlenmeyer flask.
Heat in microwave until mixture can be dissolved.
CRITICAL: Do not let the mixture boil over and out of the flask. Typical heating time for 50mL in a 2.45GHz microwave oven at full power is 30s. USE HEAT GLOVES
Gently swirl until well mixed and gently swirling periodically until ~55°C.
Gently pour molten agarose gel into housing, avoiding air bubbles.
Place desired well comb in desired position.
Once gelled, carefully remove well comb in a uniform fashion.
Remove gel mold and place in parallel direction to flow
CRITICAL: the deposit line has to be at the anode (negative pole)
Fill gel box with 0.5 xTAE until the gel is well covered.
Place the ladder on the gel, the native and digested plasmid (write down the gel map)
TIP: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffer from entering the tip.
Run the electrophoresis for 20-30min at 100V until the dye line is approximately 80% of the way down the gel
Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
Place the gel into a container filled with 100 mL of TAE running buffer and 5 μL of EtBr, place on a rocker for 20-30 mins, r
Place the gel into a container filled with water and destain for 5 mins.
Reaveal under UV lamp, visualize your DNA fragments
Miniprep
Introduction
This protocol was taken from the ThermoScientific GeneJET Plasmid Miniprep Kit. Safety: Both the Lysis Solution and the Neutralization Solution contain irritants. Wear gloves when handling these solutions.
Procedure
Note: All steps should be carried out at room temperature. All centrifugations should be carried out in a microcentrifuge at ≥ 12 000 x g (10 000-14 000 rpm, depending on the rotor type).
Be sure that the concentrated solutions have been diluted with the appropriated buffer
Pick a single colony from a freshly streaked selective plate to inoculate 5mL of LB medium supplemented with the appropriate selection antibiotic.
Incubate for 12-16 hours at 37°C while shaking at 200-250 rpm
Centrifugate the bacterial culture, >12 000 g in a microcentrifuge for 2 minutes at room temperature. Repeat until there is no more media.
Add to the pelleted cells:
250 μL of Resuspension Solution and vortex
250 μL of Lysis Solution and invert the tube 4-6 times. WAIT 2 min
350 μL of Neutralization Solution and invert the tube 4-6 times.
Lysis buffer must be neutralized before 5 minutes
Centrifuge 5 minutes.
Transfer the supernatant to the Thermo Scientific GeneJET Spin Column. Centrifuge 1 minute
Add 500 μL of Wash Solution and centrifuge for 60 s and discard the flow-through
Repeat step 5.
Centrifuge empty column for 1 minute.
Dry for 5 minutes
Transfer the column into a new tube.
Add 30 μL of Elution Buffer to the column and incubate 2 minutes.
Thaw and resuspend the T4 DNA Ligase Buffer at room temperature (10X)
In a microcentrifuge tube on ice:
Note: the table shows a ligation using a molar ratio of 1:10 vector to insert for the indicated DNA sizes./!\ T4 DNA Ligase should be added last. Note : usually put 1 μl of vector vs. 3 μl of insert DNA
Gently mix the reaction by pipetting up and down and microfuge briefly.
For cohesive (sticky) ends, incubate at room temperature for 20 minutes.
For blunt ends or single base overhangs, incubate at room temperature for 2 hours
Heat inactivate at 65°C for 10 minutes.
Chill on ice and transform 5 μl of the reaction into 50 μl competent cells.
Ligation with T4 DNA Ligase (M0202)
Introduction
Please see the NEB website for supporting information on this protocol.
Note: T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes. Use NEB calculator to calculate molar ratios.
Thaw the T4 DNA Ligase Buffer and resuspend at room temperature. Tip: Alicuote the 10x buffer less concentrated so when thawing, the DTT gets soluble more easily.
Set up the following reaction in a microcentrifuge tube on ice:
Component
Volume (µL)
10X T4 DNA Ligase Buffer
2
Vector DNA: 50 ng (0.020 pmol)
Insert DNA: 37.5 ng (0.060 pmol)
Nuclease-free water
17
T4 DNA Ligase
1
Total
20
Gently mix the reaction by pipetting up and down and microfuge briefly.
For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours.
Heat inactivate at 65 degrees C for 10 minutes.
Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. Use 25 uL DH5α cells, and add 2 uL of reaction mixture.
Transformation
Introduction
We used Subcloning EfficiencyDH5α Competent Cells from thermofisher for transforming our cells. This protocol is based on the Thermofisher protocol for these cells.
Procedure
Thaw on ice one tube of DH5αTM cells. Place 1.5 ml microcentrifuge tubes on wet ice.
Gently mix cells with the pipette tip and aliquot 50 μl of cells for each transformation into a 1.5 ml microcentrifuge tube.
Refreeze any unused cells in the dry ice/ethanol bath for 5 minutes before returning to the -80°C freezer. Do not use liquid nitrogen.
Add 1 to 5 μl (1-10 ng) of DNA to the cells and mix gently. Do not mix by pipetting up and down.
Incubate tubes on ice for 30 minutes.
Heat shock cells for 20 seconds in a 42°C water bath without shaking.
Place tubes on ice for 2 minutes.
Add 950 μl of pre-warmed medium of choice to each tube.
Incubate tubes at 37°C for 1 hour at 225 rpm.
Spread 20 μl to 200 μl from each transformation on pre-warmed selective plates. We recommend plating two different volumes to ensure that at least one plate will have well-spaced colonies.
Store the remaining transformation reaction at +4°C .Additional cells may be plated out the next day, if desired.
Incubate plates overnight at 37°C.
Transformation (RbCl-method)
Introduction
This protocol was given by Stéphanie. The aim is to make yourself Top10 competent cells.
Materials
2 * Steri cup 250mL
TrisEDTA
Procedure
Media and Solutions
500 mL LB
200 mL TFB1:
0.59 g KOAc (Cf=30 mM)
2.42 g RbCl (100 mM)
0.29 g CaCl2 2H2O (10 mM)
1.98 g MnCl2 4H2O (50 mM)
30 g Glycerol (15% wt/vol)
Adjust to pH 5.8 with 0.2 M acetic acid (do not adjust pH with KOH). Add dH2O to 200 mL. Filter sterilize. Store refrigerated at 4°C.
200 mL TFB2:
0.42 g MOPS (10 mM)
2.21 g CaCl2 2H2O
0.24 g RbCl (10 mM)
30 g Glycerol (15% wt/vol)
Adjust to pH 6.5 with KOH. Add dH2O to 200 mL. Filter sterilize. Store refrigerated at 4°C.
Preparation of Competent Cells
Streak cells from frozen stock onto LB plate. Incubate O/N at 37°C.
Pick a single fresh colony to inoculate 5 mL of LB medium. Grow O/N at 37°C. Do not vortex cells at any time after this point in the procedure.
Dilute 1 mL of culture into 50 mL LB medium prewarmed to 37°C. Grow at 37°C for 2 hours with agitation. Volumes can be scaled up 5X and all of the 5 mL overnight culture can be used.
Transfer culture to sterile 50 mL tube. Chill on ice 10-15 minutes.
Centrifuge for 10 mintutes at 2000 rpm at 4°C. Immediately aspirate off all the supernatant. Do not allow cells to warm above 4°C at any time in this procedure.
Resuspend cells in 10 mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass of plastic pipette.
Incubate cells on ice for 5 minutes.
Repeat step 8
Resuspend cells in 2 mL of ice-cold TFB2 with gentle re-pipetting. Use micropipet tip (plastic).
Incubate cells on ice for 15 minutes. Cells may be used for transformation or frozen. To freeze: aliquot cells 100 µL volumes into prechilled 0.5 mL microcentrifuge tubes (on ice). Freeze immediately on dry ice. Stire cells frozen at -80°C.
Transformation of competent cells
If starting with frozen competent cells, warm tube/cells by gently twirling between your fingers until just thawed (i.e., at ~0°C). Then, immediately place on ice for about 5 minutes.
Set up transformation as follows:
Add to 15 mL plastic round bottom tube on ice:
0-9 µL TE (Tris 10mM + EDTA 1mM)
1-10 µL DNA (10-100 ng)
10 µL final volume → /!\ 10% max of the cell competent volume
Add 100 µL of competent cells and mix by gentle repipetting. This method can be scaled down 2- to 4-fold. The maximum volume of DNA should be ~1/10 volume of cells and the maximum mass should be <= 100 ng of DNA for 100 µL of cells.
Incubate cells on ice for 20-30 minutes.
Heat shock the cells exactly 90 seconds at 42°C.
Return cells on ice 2 minutes.
Add 1 mL of LB medium. Incubate at 37°C for 45-60 minutes with slow gentle shaking. For blue/white color selection, spread IPTG and X-gal on plates now and hold at 37°C until use
Plate 0.1 - 0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic (adn IPTG and X-gal if needed). Incubate overnight at 37°C. Place at 4°C to store and/or enhance blue color. Note: The next day, liquid cultures of the transformants can be left 8 hours before the miniprep. In the best-case scenario, do the liquid culture at 8am and do the miniprep at 4pm.
Testing competent cells
Transform 100 µL of cells with 1 µL (10 pg) of pUC19 monomer (0.01 µg/µL).
Plate 0.25 mL of transformation mixture. Incubate overnight at 37°C.
Count CFU and calculate efficiency. Efficiency =# of colonies per µg =# of colonies X4 X 105. You should obtain 1-5 X 107/µg from competent cells after one freeze-thaw cycle.
1 μL 10X buffer (most enzymes can be used in Cutsmart buffer, check on NEB website)
1U enzyme pour 1 μg ADN (0,5 μL for 1 μg DNA)
H2O qsp 10 μL
heating plate
For preparative digestion:
Keep the same proportions and scale up for 30µL of DNA on 100µL final
If cut by the same Enzyme, please prepare a MIX with n+1 (n = number of sample)
For gel migration, add 2 μL of loading dye for each 10 μL mix
Procedure
Mix all the elements
Incubate 1h at enzyme specific temperature (usually 37°C)
Check if heat inactivation is required and do it accordingly /!\ if inactivation is done at high temperature put on ice after inactivation and then centrifuge to keep the evaporated water.
Electroporation
Introduction
Protocol from Lin-Cereghino, J., Wong, W., Xiong, S., Giang, W., Luong, L., Vu, J., Johnson, S. and Lin-Cereghino, G. (2005). Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. BioTechniques, 38(1), pp.44-48.
Materials
ice
linearized plasmid (with AvrII)
competent cells from the protocol cell preparation
electroporation apparatus
1.0M sorbitol
YPD
plates with gradient of zeocin
Procedure
Mix approximately 4-8μL (50–100 ng) of dialysed linearized plasmid DNA with 40 μL of competent cells in an electroporation cuvette
Incubate for 2 min on ice
Pulse 1500V, 25μF, 200Ω (You should have a Ꞇ between 4 and 5 ms. If it is >5ms, there were too many ions in the mix. It can kill cells.) (was done previously with 1500V, 10μF, 600Ω -> worked)
Resuspend immediately samples in 0.5 mL 1.0 M sorbitol and 0.5 mL YPD, incubate in a 30°C shaker for 1h30, and then plate on media containing increasing concentrations of zeocin (100, 250, 500, or 1000 μg/mL) for the selection of multicop
