Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/12/17
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Performing Type IIS assembly on the amplified, BsmBI-tagged parts.
These are the parts I currently have:
All parts are located in the "Ben's luc replacement" box on the second shelf in the large -20 °C freezer. Parts are taped together.
Overview of assembly can be found on Benchling: https://benchling.com/s/sICuMhHj/edit
Gel Extraction of HPK:CFP fragment
Perform standard gel extraction on the HPK:CFP fragment in the PCR tube. Use the entire tube contents (~45 µL) to maximize yield, if possible. The band you are aiming to extract is 2000 bp in size. It will probably be the smaller of two main bands - the larger one is ~1500 bp in size. Quantify on the plate reader.
Type IIS Assembly
We want a 2:1 molar ratio of inserts to vector, and will be using 75 ng of vector total for this reaction. Therefore the required volumes from the parts are:
Calculating volume of insert required: 2*(insert size in bp / vector size in bp) * 75 ng / insert concentration in ng/µL
Set up the reaction as follows:
Thermal cycler program:
Follow the steps for traditional transformation of chemically competent cells with ligation product. Use entire reaction volume to transform.