Team:Ionis Paris/19 10 16
Double digestion of pSB1C3-RFP and X2 by EcoRI and SpeI for the subsequent ligation of X2 in pSB1C3. pSB1C3-RFP 3 : 104.14 ng/µL (from miniprep 19/07) X2: Digestion of 75 ng (ratio 1:1 = 21.67 ng needed, ratio 2:1 = 43.33 ng needed —> 65 ng needed) In a 1.5 mL Eppendorf tube, adding in the respected order (bigger volume first and enzyme last) : NB: The digestion were done in 20 µL. Short Spin Centrifugation Incubation 1h at 37°C Store at 4°C before gel electrophoresis and purification 1% Agarose gel: Put 1 g of agarose low melting point + 100 mL of TAE 1X in a bottle of 500 mL. Mix and heat it 2 min 30 s in the microwaves. Wait the cooling of the bottle until it is tepid. Add 3 µL of Gel Red 10,000 X (0.3 X final). Flow the gel and place the combs. Wait until it is solidified. Remove slowly the combs. Drop-off: Short Speed centrifugation of samples. Addition of 4 µL of Purple loading dye 6X in the 20 µL of sample. Drop-off 10 µL of Purple ladder and 24 µL of sample. Run at 90 V. QIAquick PCR purification kit (qiagen, 28106), according to the protocol given by the supplier (available here) Run at 90 V. QIAquick Gel purification kit (Qiagen, 28704), according to the protocol given by the supplier (available here) Excise the DNA fragments from the agarose gel. Gel slice Weigh = 162mg Add 3 volumes Buffer QG (486µL) to 1 volume of gel. Incubate at 50°C for 10 min until the gel slice has completely dissolved. Vortex the tube every 2–3 min to help dissolve gel. The color of the mixture is yellow. Add 1 gel volume isopropanol to the sample and mix. Load 800 µL of each samples to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through. Load the rest and spin again. Add 500 µL Buffer QG. Centrifuge for 1 min at 13,000 rpm and discard flow-through. Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard flow-through. Centrifuge once more for 1 min at 13,000 rpm. Place QIAquick column into a clean 1.5 mL microcentrifuge tube. Add 30 µL Buffer EB to the center of the QIAquick membrane, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm. Store the purified DNA at 4°C before the ligation. QIAquick PCR purification kit (qiagen, 28106), according to the protocol given by the supplier (available here) Add 5 volumes Buffer PB (100 µL) to 1 volume of the sample (20 µL) and mix. The color of the mixture is yellow. Load the sample to the QIAquick column. Centrifuge for 1 min at 13,000 rpm and discard flow-through. Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard flow-through. Centrifuge once more for 1 min at 13,000 rpm. Place each QIAquick column in a clean 1.5 mL microcentrifuge tube. Add 30 µL Buffer EB to the center of the QIAquick membrane, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm . Store the purified DNA at 4°C before the ligation. Expected results / Obtained results: The digestion of pSB1C3-RFP was efficient, we get 2 strips at the end of the electrophoresis. The strip at 2070 pb was the digested pSB1C3 that we purified for the subsequent ligation. Ligation of X2 in pSB1C3 in order to obtain BBX2 for subsequent transformation and creation of a stock of bacteria.
The molar ratios for the ligation were calculated using NEB BioCalculator (available here) pSB1C3-RFP 3 : 2.17 ng/µL (65 ng/ 30 µL) In the following order, add : NB: The ligations were done in 30 µL for ratio 1:1 and 40 µL for ratio 2:1. Mix by pipetting Incubate for 1h at room temperature The objective is to transforme competent DH5⍺ cells with the ligation products BBX2. 5 aliquot of 100 µL DH5⍺ competent cells (from the 20/09/16). Plasmid DNA : Ligation product BBX2. Petri dish LB+Cm: Cm concentration = 25 µg/mL The objective is to transforme competent DH5⍺ cells with the ligation products BBP2mut, BBX2, BBC4, BBG2. 5 aliquots of 100 µL DH5⍺ competent cells (from the 20/09/16). Plasmid DNA : Ligation product BBP2mut, BBX2, BBC4, BBG2. Petri dish LB+Cm: Cm concentration = 25 µg/mL. —> We need 6 LB+Cm plates + 14 LB plates Thaw tubes of DH5⍺ competent cells on ice for 10 min. Mix gently and carefully pipette 50 µL of cells into the 4 transformation tubes on ice. Add the 30 µL / 40 µL plasmid DNA to the cell mixture. Carefully flick the tubes 4-5 times to mix cells and DNA. Do not vortex. Place on ice for 30 min. Do not mix. Heat shock at exactly 42°C for 45 s. Do not mix. Place on ice for 5 min. Do not mix. Pipette 250 µL of room temperature SOC into the mixture. Place at 37°C for 1h at 250 rpm. Warm selection plates to 25°C. Mix the cells thoroughly by flicking the tubes and inverting. Spread the corresponding volume onto each plate. Incubate all the plates O/N at 37°C. Expected results : Some colonies on the petri dishes LB+Cm plated with 50 µL of bacteria transformed with the different ligation products and more on the petri dishes LB+Cm plated with 200 µL of bacteria.
A bacterial lawn on the LB petri dishes without antibiotic. Obtained results: (Waiting for results) (Waiting for results) Purification of BB2mut, BBC4 and BBG2 plasmids extracted from bacterial mini-cultures. 7 Mini-cultures of bacteria transformed with BB2mut (1,2,4), BBC4 (2,4,6) and BBG2 (4) realized the 18/10 (put a colony with satisfying PCR results in 5 mL LB+Cm into a 50 mL Falcon tube). The miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104) and following the protocol given by the supplier (available on https://www.qiagen.com/fr/resources/resourcedetail?id=331740ca-077f-4ddd-9e5a-2083f98eebd5&lang=en). Miniprep Divide each 4.5 mL bacterial O/N mini-cultures into 4 Eppendorf tubes and centrifuge all those tubes at 9,000 rpm for 3 min at room temperature. Discard the supernatant. Resuspend the pellet in 62.5 μL Buffer P1 and pool the 4 Eppendorf tubes into a unique tube. Add 250 μL Buffer P2 and mix by inverting the tube 6 times. The solution turns blue. Add 350 μL Buffer N3 and mix by inverting the tube 6 times. The solution turns colorless. Centrifuge for 10 min at 13,000 rpm. Load 800 μL supernatant from step 5 to the QIAprep 2.0 spin column. Centrifuge for 1 min and discard the flow-through. Add 500 µL Buffer PB. Centrifuge for 1 min at 13,000 rpm and discard the flow-through. Add 750 µL Buffer PE. Centrifuge for 1 min at 13,000 rpm and discard the flow-through. Centrifuge once more for 1 min at 13,000 rpm. Place the QIAprep 2.0 spin column in a clean 1.5 mL microcentrifuge tube. Add 50 μL Buffer EB to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm. Store the purified DNA at -20°C. Bacteria storage : Add 100 µL of glycerol 50% to 100 µL of transformed bacteria in clean microcentrifuge 1.5 mL Eppendorf. Store at -80°C.
Digestion: pSB1C3 and X2
Objectives
Materials
Stock concentrations:
X2: ~ 150 ng/µL (from PCR 17/10)Quantity of DNA required for the ligation of C5 into BB1:
pSB1C3-RFP: Digestion of 104 ng (25 ng needed per ratio —> 50 ng of digested pSB1C3 needed —> 75 ng of pSB1C3-RFP needed) Protocol
Digestion:
Electrophoresis for digested pSB1C3-RFP:
Gel purification for digested pSB1C3:
PCR purification :
PCR purification for digested X2:
Results
Interpretation
Ligation of X2 into pSB1C3:
Objective
Materials
Concentrations of the different components after digestion and PCR purification :
X2: 2.5 ng/µL (75 ng / 30 µL) Protocols
Transformation: competent DH5⍺ cells with ligation product of BBX2
Objective
Materials
Concentrations of the different components after digestion and PCR purification :
Protocol
Materials
Protocol
Experimental conditions realized :
Transformations protocol:
Results (obtain the 20/10)
No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control).
Interpretation
Miniprep: on DH5⍺ transformed with BB2mut, BBX2, BBC4 and BBG2
Objective
Materials
From those mini-cultures, take 500 µL to realize a glycerol stock of tranformed bacteria. The 4.5 mL remaining will serve for the miniprep. Protocol
- 9 tubes of BB2mut (3 per mini-cultures)
- 9 tubes of BBC4 (3 per mini-cultures)
- 3 tubes of BBG2 (3 per mini-cultures)
