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:

Part name Gel verified? Seq verified?
pSB1A3 yes no
HA1 yes yes
EM7:ZeoR yes yes
HPK:CFP:CFP:NLS:Stop no no
HA2 yes yes

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)
pSB1A3 2189 31.4 2.39 -
HA1 134 49.6 - 0.19
EM7:ZeoR 470 132.7 - 0.24
HPK:CFP:CFP:NLS:Stop 2000 unknown - unknown
HA2 129 72.2 - 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)
pSB1A3 2.39
HA1 0.2
EM7:ZeoR 0.3
HA2 0.1
10x T4 Ligase Buffer 1
T4 Ligase (NEB) 1
BsmBI 0.5
dH2O 4.51
Total 10

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.