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), with major revisions as suggested by Dr. Haynes. Remember to use one negative control (water), one negative control (digested 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. ✓ PCR Clean-up of insert (works on restriction digests, too).
  3. Gel purification of backbone (to remove the old insert).
  4. Quantification using Take3 plate (nanodrop).
  5. Ligate using NEB T4 DNA ligase & Roche ligation buffer.
  6. Transform DH5α-Turbo with ligated plasmid. Use negative control (water), negative control (digested 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 DBN001 (gBlock) and the vector using the following reagents. Use plenty of DNA for this step; the objective is to have an abundance of digested material to work with downstream.

Reagent Volume (DBN001) Volume (mCh_pSB1A3)
DNA 10 µL 20 µL
10x FastDigest Buffer 3 3
EcoRI 1 1
SpeI 1 1
Water 15 5
Total 30 30

Mix and incubate at 37°C for 15 minutes. Skip heat inactivation (you'll be purifying the restriction digest products).

--'PCR' Clean-up of digested DBN001--

Follow the instructions in the Sigma PCR clean-up kit.

--Gel purification of pSB1A3 backbone--

Load entire volume of digest into one lane of the gel.

Gel shows three bands: one at approx. 1000bp, one at approx 2000bp and one at over 3000bp (brighter). 3000bp fragment is probably incomplete digestion of plasmid, either single cut or uncut. Excised 2000bp band (backbone without insert) and proceeded to gel purification.

Mass of band fragment excised from gel: 0.254mg

Follow the instructions in the Sigma gel extraction kit.

--DNA Quantification--

Sample ID Conc. (ng/uL) St Dev (ng/uL) 260/280
DBN001 23.96 1.373 1.719
pSB1A3 14.73 0.3239 1.692

--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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