July 20th 2016

Digestion : pSB1C3-RFP, P1, P2 and P3

Objectives

Double digestion of P1, P2, P3, and pSB1C3-RFP by EcoR1 and Pst1, for the subsequent ligation of our 3 parts in pSB1C3, in order to construct BB1, BB2 and BB3.

Materials

Stock concentrations:

pSB1C3-RFP 2 (from mini prep 19/07) —> 95.204 ng/µL

P1 : 18.12 ng/µL (from PCR purification 16/06)

P2 PCR : 16.82 ng/µL (from PCR purification 28/06)

P3 PCR : 24.64 ng/µL (from PCR purification 05/07)

Quantity of DNA required for ligation of P1, P2 and P3 into pSB1C3:

pSB1C3-RFP: 3 Digestion of 190 ng (25 ng needed per ratio —> 75 ng needed per part)

P1: Digestion of 100 ng (37.5 ng of P1 needed)

P2: Digestion of 150 ng (127.5 ng of P2 needed)

P3: Digestion of 100 ng (90 ng of P3 needed)

Protocol

Digestion:

In a 1.5 mL Eppendorf tube, adding in the respected order (bigger volume first and enzyme last) :

NB : The digestion were done in 10 µL for pBS1C3 and the P1 and P3 and 15 µL for P2.

Short Spin Centrifugation
Incubation 1h at 37°C
Store at 4°C before gel electrophoresis and purification

Electrophoresis for digested pSB1C3:

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 2 µL of Purple loading dye 6X in the 10 µL of sample.
Drop-off 10 µL of Purple ladder and 12 µL of sample.

Run at 90 V.

Gel purification for digested pSB1C3:

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 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.

PCR purification for digested P1, P2 and P3:

QIAquick PCR purification kit (qiagen, 28106), according to the protocol given by the supplier (available here)

Add 5 volumes Buffer PB (50 µL/100 µL) to 1 volume of the sample (10 µL/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.
Calculate the quantity of DNA with the Nanodrop.
Store the purified DNA at 4°C before the ligation.

Results

Electrophoresis:

Expected results / Obtained results:

Interpretation

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.