Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/12/17

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Overview

Performing Type IIS assembly on the amplified, BsmBI-tagged parts.

These are the parts I currently have:

Part name Gel verified? Seq verified?
pSB1A3yesno
HA1yesyes
EM7:ZeoRyesyes
HPK:CFP:CFP:NLS:Stopnono
HA2yesyes

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:

Part name size (bp) Conc. (ng/µL) Vol. for 75 ng (µL) Vol. for 2:1 ratio (µL)
pSB1A3218931.42.39-
HA113449.6-0.19
EM7:ZeoR470132.7-0.24
HPK:CFP:CFP:NLS:Stop2000unknown-unknown
HA212972.2-0.12

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:

Reagent Volume (µL)
pSB1A32.39
HA10.2
EM7:ZeoR0.3
HPK:CFP:CFP:NLS:Stop??
HA20.1
10x T4 Ligase Buffer1
T4 Ligase (NEB)1
BsmBI0.5
dH2O4.51
Total10

Thermal cycler program:

  • 45 °C, 2 min
  • 16 °C, 5 min
    • Repeat the steps above 30x
  • 60 °C, 10 min
  • 80 °C, 20 min
  • 4 °C, ∞

Bacterial Transformation

Follow the steps for traditional transformation of chemically competent cells with ligation product. Use entire reaction volume to transform.


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