Haynes Lab:Notebook/CRISPR Editing/2015/01/23: Difference between revisions

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'''Topo cloning'''
'''Topo cloning'''
*purify uncut band from agarose gel
*purify uncut band from agarose gel
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample Read#'''
| align="center" style="background:#f0f0f0;"|'''260'''
| align="center" style="background:#f0f0f0;"|'''280'''
| align="center" style="background:#f0f0f0;"|'''260/280'''
| align="center" style="background:#f0f0f0;"|'''ng/µL'''
|-
| g033||0||-0.002||-0.267||0.423
|-
| g034||0.004||0.003||1.281||4.288
|}
*add 'A's to the ends of the uncut products
*add 'A's to the ends of the uncut products
:From the manual:
:From the manual:

Revision as of 16:22, 23 January 2015

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01/23/2015

Sent out untreated Luc14 gDNA PCR product of P197/P198 for sequencing:

Label Primer Direction
RD-1-3 P175 F
RD-4-6 P161 F
RD-7-9 P173 F
RD-10-12 P159 F
RD-13-15 P170 R
RD-16-18 P156 R
RD-19-21 P172 R
RD-22-24 P158 R



Topo cloning and quantification of editing efficiency with restriction enzyme'

cutting 1μg of untreated and g033 and g034 treated Luc14 cells gDNA PCRd with P197/P198 and column purified samples from 14.12.06 experiment with PfoI at 37°C for 3 hours.

Sample Read# ng/µL ul for 1ug ul H2O ul buffer ul PfoI
Untreated 85.402 11.71 5.29 2 1
g033 78.942 12.67 4.33 2 1
g034 80.841 12.37 4.63 2 1

Quantification with 1% agarose gel




Topo cloning

  • purify uncut band from agarose gel
Sample Read# 260 280 260/280 ng/µL
g033 0 -0.002 -0.267 0.423
g034 0.004 0.003 1.281 4.288
  • add 'A's to the ends of the uncut products
From the manual:
After purification, add Taq polymerase buffer, dATP, and 0.5 unit of Taq polymerase. Incubate the reaction for 10–15 minutes at 72°C and use in the TOPO® Cloning reaction.
  • Set up the TOPO Cloning reaction
Mix the reaction below gently and incubate for 5 minutes at room temperature (22°C to 23°C):
Reagent* Volume
Fresh PCR product 0.5–4 µL
Salt Solution 1 µL
Water add to a total volume of 5 µL
TOPO® vector 1 µL
Final Volume 6 µL


Notes from the manual:
Note: The red or yellow color of the TOPO® vector solution is normal and is used to visualize the solution.
A Salt Solution (1.2 M NaCl; 0.06 M MgCl2) is provided to adjust the TOPO® Cloning reaction to the recommended concentration of NaCl and MgCl2... We have found that including salt (200 mM NaCl, 10 mM MgCl2) in the TOPO® Cloning reaction can increase the number of transformants 2- to 3-fold. In addition, incubating the reaction mixture for greater than 5 minutes in the presence of salt can also increase the number of transformants... Including salt in the TOPO® Cloning reaction allows for longer incubation times because it prevents topoisomerase I from rebinding and potentially nicking the DNA after ligating the PCR product and dissociating from the DNA. The result is more intact molecules, leading to higher transformation efficiencies.
For most applications, 5 minutes will yield sufficient colonies for analysis. Depending on your needs, the length of the TOPO®-cloning reaction can be varied from 30 seconds to 30 minutes. For routine subcloning of PCR products, 30 seconds may be sufficient. For large PCR products (greater than 1 kb) or if you are TOPO®-cloning a pool of PCR products, increasing the reaction time will yield more colonies.
You may store the TOPO® Cloning reaction at −20°C overnight.


Place the reaction on ice and proceed to Transform One Shot® competent cells
  • Transform


Note: It is essential that LB plates containing ampicillin are pre-warmed prior to spreading. 1. Add 4 µL of the TOPO® Cloning reaction from Set up the TOPO® Cloning reaction, step 2 on page 13, into a vial of One Shot® chemically competent E coli and mix gently. Do not mix by pipetting up and down. 2. Incubate on ice for 5 minutes. 3. Spread 50 µL of cells on a prewarmed LB plate containing 50–100 µg/mL ampicillin and incubate overnight at 37°C. 4. An efficient TOPO® Cloning reaction should produce several hundred colonies. Pick ~10 colonies for analysis (see Analyze positive clones on page 20).




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