Manual preparation of solutions for spotting DNA by contact printer (for reverse transfection)

Authors

Beate Neumann, Alen Piljic

Summary

This is a protocol for preparing plasmid DNA for spotting in dishes by contact printer and reverse transfection.

• Image 1 Spotted LabTek dish.

Procedure

Prepare the following solutions fresh for each experiment

1. 0.2% gelatine

• dissolve 0.2g gelatine in 100ml H2O
• heat to 56°C for 20 min in waterbath for dissolving
• cool down before use
• filter with 0.45μm pore filter

2. sucrose/Opti-MEM solution

• 1.37g sucrose in 10ml Opti-MEM dissolve w/o shaking on bench

Prepare DNA/transfection reagent-fibronectin/gelatine mix in the following order

1. 3µl sucrose/Opti-MEM

2. 3.5µl Lipofectamine 2000; mix 8x by pipetting

3. 5µl DNA (400ng/µl); mix 8x by pipetting

→ incubate 20min at RT

4. 7.25µl 0.2% gelatine (supplemented with final 0.001% fibronectin); mix 8x by pipetting

Total volume = 18.75 µl

The transfection solution can now be printed on dishes (e.g. LabTek chambered coverglass) using the contact printer ChipWriter Pro (Bio-Rad). After spotting, dishes should be placed in a box containing drying pearls and stored at RT for at least 24 h before cell seeding.

You can find cell seeding protocol here.

Further details

The protocol was provided by B. Neumann.

References

Modified from:

Erfle, H., Neumann, B., Liebel, U., Rogers, P., Held, M., Walter, T., Ellenberg, J., and Pepperkok, R. (2007) Reverse transfection on cell arrays for high content screening microscopy. Nat. Protoc. 2, 392–399.

Stats

Post a comment

You need to be signed in to post comments. You can sign in here.

Comments

There are no comments yet.