Team:Ionis Paris/Notebook/06 10 2016
Purification of BBC2 plasmid extracted from bacterial mini-cultures in order to have a stock of this plasmid and to sequence it. 5 Mini-cultures of bacteria transformed with BBC2 (1,4,7,8) realized the 05/10 (put a colony with satisfying PCR results in 5 mL LB+Cm into a 50 mL Falcon tube).
From those mini-cultures, take 300 µL to realize a glycerol stock of tranformed bacteria. The 4.7 mL remaining will serve for the miniprep. The miniprep were realized using the QIAprep® Spin Miniprep Kit (Qiagen, ref: 27104) 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. Calculate the quantity of DNA with the Nanodrop. Store the purified DNA at -20°C. 1. Add 100 µL of glycerol to 100 µL of transformed bacteria in a clean 1.5 mL Eppendorf tube. 24 tubes of BB2 3 tubes of BB3 12 tubes of BB12 Double digestion of BB12 by SpeI and PstI, and of BBX4 by XbaI and PstI for the subsequent ligation of X4 in BB12 in order to create BBX5. BB12mut-4: 250 ng/µL (from mini prep realised the 07/09) BBX4-1: ~150 ng/µL (from mini prep realised the 03/10) BB12: Digestion of 100 ng (50 ng needed per ratio) BBX4: Digestion of 270 ng (ratio 3:1 = 24.61 ng of digested X4 needed, ratio 5:1 = 41.02 ng of digested X4 needed —> 65.63 ng of X4 needed —> 255.38 ng of BBX4 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 2min 30s in the microwaves. Wait the cooling of the bottle until it is tepid. Add 3 µL of Gel Red 10,000X (0.3X 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 25 µL of sample. 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 = 304 g Add 3 volumes Buffer QG (612 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 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. Store the purified DNA at 4°C before the ligation. Expected results / Obtained results: The digestion of BBX4 was not efficient, we get 1 very light strip at the end of the electrophoresis. The strip that we obtained correspond to the backbone. Considering the low intensity of the backbone strip, we can suppose that the second strip of 717 bp, corresponding to the digested X4, is too low to be observable. We can conclude that the quantity of DNA was not enough, the stock concentration was overestimated. We have to make a new digestion using a bigger quantity of BBX4. The objective is to transforme competent DH5⍺ cells with the ligation products BB-B0015. —> We need 6 LB+Cm plates + 4 LB plates Expected results: Some colonies on the petri dishes LB+CHL plated with 1 µL of bacteria transformed with the different ligation products, more on the petri dishes plated with 10 µL, and more on the petri dishes plated with 100 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 only 2 colonies on the petri dish plated with 100 µL of transformed bacteria and none in the two others, plated with 10 µL and 1 µL. The transformation worked. Colonies contain a plasmid with the Chloramphenicol resistance gene, present in pSB1C3.
Miniprep: on DH5⍺ transformed with BBC2
Objectives
Materials
Protocol
Miniprep:
Bacteria storage :
Digestion: BB12 and BBX4
Objectives
Materials
Stock concentrations:
Quantity of DNA required for ligation of P1, P2 and P3 into pSB1C3:
Protocol
Digestion:
Electrophoresis for digested BBX4:
PCR purification for digested BB12:
Results
Electrophoresis:
Interpretation
Transformation: competent DH5⍺ cells with BB-B0015
Objectives
Materials
Protocol
Experimental conditions realized:
Transformation Protocol:
Results (obtained on the 07/10)
Interpretation
