Haynes Lab:Notebook/CRISPR Editing/2015/01/29: Difference between revisions
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==01/29/2015== | ==01/29/2015== | ||
'''Topo cloning''' | |||
Added dATPs to the ends of untreated and g034 treated Luc14 P197/198 PCR products | Added dATPs to the ends of untreated and g034 treated Luc14 P197/198 PCR 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. | |||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''thing''' | | align="center" style="background:#f0f0f0;"|'''thing''' | ||
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Incubated at 72°C for 15 min in PCR machine | Incubated at 72°C for 15 min in PCR machine | ||
<br> | <br> | ||
Cut with PfoI for 3 hours at 37°C in heat block | Cut with PfoI for 3:12 hours at 37°C in heat block | ||
{| {{table}} | {| {{table}} | ||
| align="center" style="background:#f0f0f0;"|'''thing''' | | align="center" style="background:#f0f0f0;"|'''thing''' | ||
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| PfoI||1ul | | PfoI||1ul | ||
|} | |} | ||
<br> | |||
Heat inactivated in PCR machine, 65°C for 20 minutes. | |||
<br> | |||
*Set up the TOPO Cloning reaction | |||
:Mix the reaction below gently and incubate for 5 minutes at room temperature (22°C to 23°C) | |||
:''Decided to do two reactions for each samples, one wiht .5ul and one with 4ul of PCR product'' | |||
:{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Reagent*''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| Fresh PCR product ||0.5 or 4 µL | |||
|- | |||
| Salt Solution ||1 µL | |||
|- | |||
| Water ||add to a total volume of 5 µL | |||
|- | |||
| TOPO® vector ||1 µL | |||
|- | |||
| Final Volume ||6 µL | |||
|} | |||
<br> | |||
Incubated for 30 minutes at room temp.<br> | |||
::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. | |||
<br> | |||
:Place the reaction on ice and proceed to Transform One Shot® competent cells | |||
*Transform | |||
:warmed LB plates for 30 40 minutes | |||
:added 2ul and 4ul of each of the four reactions to 50ul of cells | |||
:incubate on ice for 10 minutes | |||
:plate | |||
:grow overnight at 37 in wang lab incubator | |||
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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Revision as of 16:42, 29 January 2015
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01/29/2015Topo cloning Added dATPs to the ends of untreated and g034 treated Luc14 P197/198 PCR products
Incubated at 72°C for 15 min in PCR machine
|