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

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Overview

Trying the ligation again (previous attempt), with major revisions as suggested by Dr. Haynes. DBN001 is the gBlock containing homology arms for insertion into Gal4-EED/Luc and Type IIS cloning sites to add HPK-CFP. pSB1A3 is a standard cloning vector for molecular biology work. The plasmid I'm using contains an mCherry insert, so I can easily recognize colonies containing the DBN001 insert by their lack of red coloration. 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) 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 insert (gBlock) and the mCh_pSB1A3 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 Insert (µL) Vector (µL)
DNA 10 µL 20
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/µL) St Dev (ng/µL) 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.

To determine amount of insert to use in the ligation reaction:

  • Mass of insert = (insert length / backbone length) * 2 * mass of backbone
  • Mass of insert = (228/2132) * 2 * 50ng
  • Mass of insert = 10.7ng

Round up to 15ng to be on the safe side.

Component Volume (µL)
2x Roche DNA Ligase Buffer 10
Vector DNA 50ng (3.39 µL)
Insert DNA 15ng (0.61 µL)
water 5
NEB T4 DNA Ligase 1
Total 20

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

Transformation

Transform cells with entire volume of ligated plasmid. Remember to use negative and positive controls (water and untreated vector, respectively).

Plates are incubating at room temperature on my bench (Bench 5). Transformation was performed on 4/22/2015 at 10am - check plates daily morning and evening for colonies.