Team:Ionis Paris/27 07 16
Double digestion of P2 by XbaI and PstI and double digestion of BB1 (pSB1C3-P1) by SpeI and PstI for the subsequent ligation of P2 in BB1 in order to obtain BB2 (pSB1C3-P1-P2). P2: 16.82 ng/µL (from PCR purification 05/07) BB1-2: 97.3 ng/µL (from mini prep 26/07) P2 : Digestion of 100 ng (ratio 2:1 = 33.07 ng ; ratio 3:1 = 49.61 ng —> 82.68 ng of digested P2 needed) BB1: Digestion of 75 ng (25 ng needed per ratio—> 50 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 purification 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 50 µL Buffer EB to the center of the QIAquick membrane, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm. Calculate the quantity of DNA with the Nanodrop. Store the purified DNA at 4°C before the ligation. Ligation of P2 into BB1 for subsequent transformation and amplification of BB12.
BB1: 97.3 ng dans 30 µL —> 3.24 ng/µL In the following order, add: NB: The ligations were done in 20 µL for the ratio 2:1 and 30 µL for the ratio 3:1 Mix by pipetting Incubate for 1h at room temperature. The objective is to transforme competent DH5⍺ cells with the ligation products BB12. 3 aliquots of DH5⍺ Competent cells (from the 23/07/16) Plasmid DNA : Ligation products BB12 (from the 27/07/16), BB2 and BB3 (from the 26/07/16) Petri dish LB+Cm: Cm concentration = 25 µg/mL We need 10 LB+Cm plates + 6 LB plates Thaw 2 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 5 µL / 15 µL / 25 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. Some colonies on the petri dishes LB+Cm plated with 100 µL of bacteria transformed with the different ligation products and more on the petri dishes LB+Cm plated with the pellet of bacteria. We obtained expected results. Our bacteria seems to be tranformed with BB12 as they are resistant to chloramphenicol. However, this resistance can be due to the plamid BB1. It is possible that BB1 has closed up on itself. Therefore a PCR colony is needed to ensure that the bacteria hold BB12. The objective is to transforme competent DH5⍺ cells with the ligations products BB2 and BB3. DH5-alpha competent cells (competent from the 23/07/16) Plasmid DNA : Ligation products BB2 and BB3 Petri dish LB+Cm: Cm concentration = 25 µg/mL We need 26 LB+Cm plates + 10 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 5 µL / 15 µL / 25 µL / 35 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. Some colonies on the petri dishes LB+Cm plated with 100 µL of bacteria transformed with the different ligation products and more on the petri dishes LB+Cm plated with the pellet of bacteria. We obtained expected results. Our bacteria seems to be tranformed with BB2 and BB3 as they are resistant to chloramphenicol. However, a PCR colony is needed to check the size of the plasmid present in colonies, and therefore in order to ensure that bacteria incorpore the correct plasmid.
Digestion: P2 and BB1
Objectives
Materials
Stock concentrations:
Quantity of DNA required for the subsequent ligation:
Protocol
Digestion:
PCR purification for P2 and BB1:
Ligation: P2 into BB1:
Objective
Different ligation ratios are going to be tested 2:1 and 3:1. The molar ratios for the ligation were calculated using NEB BioCalculator (available here)Materials
Concentrations of the different components after digestion and PCR purification:
P2: 100.92 ng dans 30 µL —> 3.36 ng/µLProtocol
Transformation: competent DH5⍺ cells with the ligation product BB12:
Objective
Material
Protocol
Experimental conditions achieved :
Results (obtain the 28/07)
Expected results :
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:
Interpretation
Transformation: competent DH5⍺ cells with ligation products BB2 and BB3
Objective
Materials
Protocol
Experimental conditions achieved :
Transformations protocol:
Results (obtain the 28/07)
Expected results :
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:
Interpretation
