Team:Ionis Paris/Notebook/04 10 2016
Double digestion of BB1 by SpeI and PstI, and of BBC5 by XbaI and PstI for the subsequent ligation of C5 in BB1. BB1-2: 97,3 ng/µL (from mini prep realised the 26/07) BBC5-1: ~175 ng/µL (from mini prep realised the 03/10) BB1: Digestion of 50 ng (25 ng needed per ratio) BBC5: Digestion of 175 ng (ratio 3:1 = 25 ng of digested C5 needed, ratio 2:1 = 16.5 ng of digested C5 needed —> 42 ng of C5 needed —> 142 ng of BBC5 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 1 h at 37°C. Store at 4°C before gel electrophoresis or 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 2min 30s in the microwaves. Wait the cooling of the bottle until it is tepid. Add 5 µL of Gel Red 10,000 X (0.5 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 each samples. Drop-off 10 µL of Purple ladder and 25 µL of each samples. Run at 90 V. QIAquick Gel purification kit (Qiagen, 28704), according to the protocol given by the supplier (available here) Excise the DNA fragment from the agarose gel. Gel slice Weigh = 0.235 g Add 3 volumes Buffer QG (705 µL) 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 BBC5 was efficient, we get 2 strips at the end of the electrophoresis. The strip at 857 pb was the digested C5 that we purified for the subsequent ligation. Ligation of C5 into BB1 in order to obtain BBC2, for subsequent transformation and creation of a stock of bacteria.
The molar ratios for the ligation were calculated using NEB BioCalculator BB1: 3.24 ng/µL (97 ng / 30 µL) In the following order, add : NB: The ligations were done in 30 µL Mix by pipetting. Incubate for 1 h at room temperature. The objective is to transforme competent DH5⍺ cells with the ligation products BBC2. 2 aliquot of 100 µL DH5⍺ competent cells (from the 20/09/16). Plasmid DNA: Ligation product BBC2. Petri dish LB+Cm: Cm concentration = 25 µg/mL We need 6 LB+Cm plates + 4 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 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.
No colonies on the LB+Cm petri dish plated with bacteria transformed with no plasmid (- control). Obtained results: We obtained expected results. We obtained more colonies with the ratio 2:1. The transformation worked. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3. However, this resistance can be due to the plamid BB1. It is possible that BB1 has closed up on itself. A PCR colonie is necessary to check the size of the plasmid present in colonies, and therefore to ensure that bacteria incorpore the correct plasmid BBC2.
Digestion: BB3 and BB12
Objectives
Materials
Stock concentrations:
Quantity of DNA required for the subsequent ligation:
Protocol
Digestion:
Electrophoresis for digested BBC5:
Gel purification for digested C5:
Competence:
PCR purification for digested BB1:
Results
Electrophoresis:
Interpretation
Ligation: P3 into BB12
Objectives
Materials
Concentrations of the different components after digesion and PCR purification :
C5: 1.67 ng/µL (50 ng / 30 µL)
Transformation: competent DH5⍺ cells with ligation product BB123
Objectives
Materials
Protocol
Experimental conditions realized :
Transformations protocol:
Results (obtained the 05/10)
Interpretation
