T7 Endonuclease I cleavage assay

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https://www.neb.com/protocols/2014/08/11/determining-genome-targeting-efficiency-using-t7-endonuclease-i

https://www.neb.com/products/e3321-engen-mutation-detection-kit

idtdna CRISPR/Cas9 editing: mutation detection.

http://www.genecopoeia.com/product/indelcheck-detection-system/

https://www.diagenode.com/files/protocols/Cas9-editing-mutation-detection-protocol.pdf

Cas9-editing-mutation-detection-protocol From DiaGenode
T7 Endonuclease I cleavage assay
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The method is pretty straightforward and consists of 4 parts: DNA isolation, PCR of desired locus, denaturation and re-annealing, and T7 endonuclease I cleavage.

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* DNA isolation *
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Use your favorite protocol, or the quick ‘n’ dirty method below

HotSHOT lysis

1. Trypsinize cells from a well of a 24-well or 12-well microfuge tube. Collect in an eppendorf, spin out the supernatant. You don’t need a lot of cells, eyeball maybe 10-20 uL cell pellet at most for efficient lysis

2. Add 75-100 µl Alkaline Lysis Reagent. Assure that the cell pellet is completely submerged.

3. Incubate at 95°C for 30-60 min.

4. Add 75-100 µl Neutralization Reagent using a new aerosol-barrier tip for each sample. Mix well, using tip to break up tissue. Some people like to centrifuge the tubes after this step and transfer the neutralized supernatant to a new tube, but this is not necessary.

5. Use 2-3 µl of neutralized supernatant per 20-25 µl PCR reaction, more and you might start to inhibit the PCR reaction (usually the case for mouse.

Alkaline Lysis Reagent

To 25 ml water, add:
62.5 µl of 10 N NaOH (final concentration is 25 mM.)
10.0 µl of 0.5 M disodium EDTA (final concentration is 0.2 mM, pH should be about 12 but should not have to be adjusted.)

Make fresh every one to two months. Keep solution at room temperature.
Neutralization Reagent

40 mM Tris-HCl pH should be about 5 but should not have to be adjusted.)

Keep solution at room temperature.

Make 1 M Tris-HCl with Tris hydrochloride salt (not the base).

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* PCR reaction *
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You preferably want to use a high-fidelity polymerase with hotstart characteristics. I used Q5 hotstart from NEB because it’s cheap and robust. Design primers of your targeted locus using Primer-BLAST; set the desired target size to be between 200-1000 bp, %GC-content 40-60%, melting temperature optimum at 60, oligo size 18-25 nt in length.

Protocol that I followed:

Component 25 µl Reaction
5X Q5 Reaction Buffer 5 µl
10 mM dNTPs 0.5 µl
10 µM Forward Primer 1.25 µl
10 µM Reverse Primer 1.25 µl
Template DNA 2 µl
Q5 Hot Start 0.25 µl
Nuclease-Free Water to 25 µl

You might need to add GC-enhancer if the region that you are PCRing up is especially GC-rich or has a GC-rich stretch of nucleotides.

Thermocycling Conditions for a Routine PCR

STEP TEMP TIME
Initial Denaturation 98°C 30 seconds
25–35 Cycles 98°C 10 seconds
*50–72°C 30 seconds
72°C 30 seconds/kb
Final Extension 72°C 2 minutes
Hold 10°C

Use the NEB calculator to calculate the annealing temperature for your oligos. If it calculates your oligos for use at 72°C, do a 2-step reaction, but extend the 72°C to 60 seconds.

I do several reactions to achieve enough substrate for the assay — 3-4 reactions generally will yield a nice quantity. You will need at least ~200 ng per reaction. Load a couple of µl from a reaction on a gel to make sure that you have a single band (also load your negative reaction to make sure that you have no contamination).

I purify using a spin column and elute in 50 µl of warm (60°C) elution buffer. This will typically yield ~20-30 ng/µl of product.

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* Re-annealing *
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Add ~200-500 ng of product to a 20 µl reaction of H2O and (2 µl) 1X NEB Buffer 2. You will need 2 tubes per reaction, one for the nuclease and one for a negative (non-nuclease) control. Denature and re-anneal in a PCR machine using the parameters below:

Temperature Time
95 °C 10 min
95 °C to 85 °C (‐2.0 °C/s)
85 °C 1 min
85 °C to 75 °C (‐0.3 °C/s)
75 °C 1 min
75 °C to 65 °C (‐0.3 °C/s)
65 °C 1 min
65 °C to 55 °C (‐0.3 °C/s)
55 °C 1 min
55 °C to 45 °C (‐0.3 °C/s)
45 °C 1 min
45 °C to 35 °C (‐0.3 °C/s)
35 °C 1 min
35 °C to 25 °C (‐0.3 °C/s)
25 °C 1 min
10 °C Hold ∞

On our machine ‐2.0 °C/s is a 50% ramp rate and a ‐0.3 °C/s is a 2% ramp rate.

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* T7 Endonuclease I cleavage assay *
************************************

To each tube add 1 µl of T7 endonuclease I (10 U). Incubate at 37°C for 15 min. Stop the reaction by the adding 2 µl of 0.5 M EDTA. Load half (or more) on a 2% TBE or 1.5% SB agarose gel. Staining afterword will give a cleaner signal-noise, in which case wait until the orange dye is near the bottom of the gel (and the cyan dye is about 1/3-1/2 the way down) before you stop and stain the gel (0.5 ug/ml of EtBr for 15 min, rinse in H2O, destain 15 min in H2O).

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UVB, UVA and glass

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UVA destroys Vitamin D in the body (many previous hints of this).
Window glass block most of UVA, but ALL of UVB – thus getting UV thru glass appears to decrease your levels of vitamin D.
This may be one of the reasons that truckers live much shorter lives:

UVA without UVB : lowering of vitamin D levels.
UVA + UVB: net increase in blood levels of vitamin D.

UVB only: larger net increase (a single hint that it might be 6x this has not been confirmed).
Some bulbs have just UVB.
There is a way to block all but UVB:i.e. block UVA and visible and IR (Heat) – see below:

http://www.vitamindwiki.com/UVA+decreased+serum+levels+of+vitamin+D+-+RCT+Oct+2012

http://www.vitamindwiki.com/Vitamin+D+from+low-cost+UVB+lamps

http://www.uvguide.co.uk/phototherapyphosphor-tests.htm

http://www.who.int/uv/health/solaruvradfull_180706.pdf

KEGG Organisms: Complete Genomes

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http://www.genome.jp/kegg/catalog/org_list.html

How to precipitate viruses ( Bacteriophage, lentivirus and …) at 13,000 g in 10 min

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Material & Reagents

The following materials and reagents are needed for the procedure:

  • A bench microcentrifuge, sterile DNase-free microtubes, aerosol-barrier tips, and a bucket of ice.

  • TBS (1x) or Tris Buffer Saline: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, sterile, made 1x from commercial 10x stock solution(prevent possible protease contaminations).

  • PEG/NaCl (5x) stock solution: PEG-8000 20%, NaCl 2.5 M. Dissolve 100 g PEG-8000 (20% w/v) and 75 g NaCl (2.5 M) in 400 ml ddH20 and bring to a final volume of 500 ml by stirring at RT. The solution can be autoclaved (optional) but mixing during the cooling period is required to prevent a phase separation. Store at RT.

Procedure

  1. Transfer 1500 µl of bacterial culture containing the phage particles to a microfuge tube.

  2. Spin down bacteria by microcentrifugation for 2 min at 13,000 g.

  3. Transfer 1200 µl of supernatant to a clean microfuge tube taking care of not touching the pellet of bacteria with the tip.

  4. Add 300 µl of PEG/NaCl 5x and mix thoroughly by inversion; do not vortex.

  5. Chill the tube on ice. After a few minutes, take the tube out; wipe it with a clean tissue and expose the supernatant to indirect lighting. By rocking the tube back and forth with the fingers, PEG-precipitated virions are often seen by the naked eyes. If a precipitate is visible, the incubation can be shortened to 5 min, otherwise continue the incubation on ice up to a full hour. Some protocols in the literature suggests just 5 min on ice. Note that long incubations may promote proteolysis, especially when epitope tags are present, and it is likely good practice to shorten the precipitation as much as possible.

  6. Pellet the virions by microcentrifugation for 3 min at 13,000 g. (Note by me: If you have no pellet, put it in -80, then repeat centrifugation).

  7. Carefully remove the bulk of supernatant with a large tip and discard in an appropriate container, taking care of not spreading bacteriophage on gloves and pipettors; microfuge again for 1 min at 13,000 g; remove all residual supernatant with a 100-µl tip and discard the tip. The second centrifugation is essential to 1) collect all the phage particles at the bottom of the tube and 2) achieve a complete removal of bacterial supernatant.

  8. Resuspend the pellet by vigorous vortexing with 120 µl of TBS (1x) (1/10th of the initial culture volume) and incubate on ice for another hour. Sometimes, it is easier to let the pellet soften for a few min before resuspending the virions. Again, the incubation time can be shortened when large amounts of virions were precipitated but a safe 30-min incubation step is recommended to prevent a loss of particles during the clearing step.

  9. Vortex vigorously again and clear the phage solution by microcentrifugation for 1 min at 13,000 g; transfer the
    phage solution to a clean microtube and proceed to virion quantification.

Troubleshooting

Low virion counts

If your phage yields are too low, e.g. less than 0.1 O.D.x mL in the original culture, most likely virions are embedded with bacterial debris, the U.V. spectrum will exhibit a shallower minimum or worst, no minimum below 269 nm, and the concentration of virions measured by this method will be erroneous. It is technically difficult to purify phage further by PEG-precipitation in this situation.

To improve phage yields and take full advantage of this technique, 1) if you are using a helper phage, make sure that the density of the bacterial culture is optimal at the time of superinfection (e.g. around 0.4-0.5 OD600 for TG1 and SS320), 2) if you are using M13KO7 as a helper phage, switch to a more efficient packager such as CM13, and 3) more importantly, try to switch to a phage display system like the pADL Phagemid Vector System where expression is under tight control. Many old phagemid systems such as pComb3 or Phen2 vectors use a very leaky lac promoter driving overexpression of the fusion protein even in absence of IPTG, resulting in bacterial toxicity, slow bacterial growth, bacterial lysis and limited phage production.

Centrifugation Table

The protocol can be easily adapted to larger scale preparations by adjusting the centrifugation time while keeping the relative centrifugal force to 13,000 g as indicated in the following table:

Preparation

mini (1-2 ml)

midi (20-50 ml)

maxi (100-400 ml)

Bacterial Clearance

2 min

5 min

10 min

Centrifugation

3 min

10 min

20 min

Clearing

1 min

5 min

10 min

From:

http://www.abdesignlabs.com/technical-resources/bacteriophage-preparation/