Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/21

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Cloning of DBN001 (gBlock insert) into mCh_pSB1A3

Trying the ligation again (previous attempt). Remember to use one negative control (water), one negative control (dephosphorylated backbone), and one positive control (untreated vector).

Major steps:

  1. Restriction digest the vector and insert with EcoRI and SpeI followed by heat inactivation.
  2. Dephosphorylate the backbone followed by heat inactivation.
  3. Ligate using NEB T4 DNA ligase.
  4. Transform DH5α-Turbo with ligated plasmid. Use negative control (water), negative control (dephos. backbone), and positive control (untreated vector).

mCherry vector concentration is 392 ng/µL. gBlock concentration is 311 ng/µL.

--Restriction Digest--

In two separate tubes, digest the gBlock and the vector using the following reagents.

Reagent Volume (DBN001) Volume (mCh_pSB1A3)
DNA 100ng (0.3 µL) 600ng (1.5 µL)
10x FastDigest Buffer 3 3
EcoRI 1 1
SpeI 1 1
Water 24.7 23.5
Total 30 30

Mix and incubate at 37°C for 15 minutes. Then heat-inactivate by incubating at 80°C for 20 minutes.

--Dephosphorylation--

Dephosphorylate the digested vector using an alkaline phosphatase.

Dephosphorylation (Roche)

Reagent Volume
DNA (clean digest) up to 17 μL (340 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
Total 20 µL

Incubate at 37°C for 10 min, then deactivate phosphatase at 75°C for 2 min. Final DNA concentration is 17 ng/μL.

--Ligation--

Ligate using Roche DNA Ligase buffer and NEB T4 Ligase.

Component Volume
10x Roche DNA Ligase Buffer 2
Vector DNA 50ng (3 μL)
Insert DNA 37.5ng (11.25 μL)
water 2.75 µL
NEB T4 DNA Ligase 1
Total 20 µL

Incubate at room temp. for 10 minutes, then heat inactivate at 65°C for 10 minutes.

--Transformation--

Set aside 2µL of ligated plasmid for gel electrophoresis. Transform cells with entire remaining volume of ligated plasmid. Remember to use negative and positive controls (water and untreated vector, respectively).



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