AliCE^® Cell-Free Protein Synthesis Kit Quick Start Guide

Important – Before you begin

• RNase contamination leads to lower or no protein yields. Only use RNase-free filter tips and wear gloves at all times!
• The ALiCE^® CFPE Kit requires oxygen during the whole reaction time for a successful reaction. Do not seal reaction vessels!

Step 1 – Choose reaction vessel

ALiCE^® Tubes or 96 half-well plate with lid

Step 2 – Prepare DNA

Thaw DNA template at room temperature, mix briefly and do not heat-treat.

Positive control plasmids pALiCE01 and pALiCE02 are supplied ready-to-use at 250 ng/µL. See instruction manual for additional information on template preparation.

Step 3 – Thaw ALiCE^® reaction mix

Thaw the ALiCE^® reaction mix in a water bath at room temperature (20 - 25 °C). Do not vortex ALiCE^® Reaction mix! Place on ice directly after thawing. Start reactions within 30 min after thawing.

Freeze remaining lysate at -80 °C. Do not use liquid nitrogen. Avoid more than one freeze-thaw cycle.

Step 4 – Reaction assembly and reaction

4a) ALiCE® Tubes
Assemble at room temperature:
Component	Volume
ALiCE® Reaction mix	48 µL
pALiCE vector / DNA template	2 µL
Total volume	50 µL
Note: Only use the supplied punctured caps to close ALiCE® Tubes.

4b) 96 half well plates*
Assemble at room temperature:
Component	Volume
ALiCE® Reaction mix	48 µL
pALiCE vector / DNA template	2 µL
Total volume	50 µL
Important: Add ~ 75 µL of water in the interwell space.

Note: When using pALiCE plasmids with different inserts, dilute or concentrate to the same final molarity of 5 nM. DNA concentration may significantly affect protein yield.

* For best results, use plates from Greiner Bio-One, Art. No. 675086

Step 5 – Reaction parameters

4a) ALiCE® Tubes
Incubate the ALiCE® tubes in an orbital table-top shaker at 700 rpm and 25 °C for 48 h. We specify a 3 mm shaking diameter for optimal results.

4b) 96 well plates
Incubate the plates in an orbital shaker at 500 rpm and 25 °C for 48 h. We specify a 25 mm shaking diameter and a controlled humidity of > 70 % for optimal results.

Due to the long incubation time, evaporation may occur. Please refill the reaction vessel with RNase-free water to 50 µL after the reaction is completed.

	Do not seal reaction vessels or plates!
	When using pALiCE01 as a positive control, a yellow color should be visible in the Reaction Mix after 48 hours.

Usage Notes

• Template DNA is a common source of RNase contamination. Purification of DNA with a procedure based on anion exchange chromatography is therefore highly recommended. Alternatively, the template DNA can be purified by phenol-chloroform extraction prior to use with the ALiCE^® kit. Also, RNase inhibitor can be added before the reaction.
• When using other reaction conditions or other volumes than stated above or hindering the oxygen transfer by sealing the plates, protein yields will be diminished.
• For additional information on plasmid preparation, protein purification (SDS PAGE) and micro- some targeting, please refer to the instruction manual.

For in vitro / research use only.

The kit is shipped frozen on dry ice, please check if the contents are still frozen upon delivery. Contact us immediately if any issues with delivery have occurred.