Haynes Lab:Notebook/CRISPR Editing/2015/02/07: Difference between revisions

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(Autocreate 2015/02/07 Entry for Haynes_Lab:Notebook/CRISPR_Editing)
 
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==02//2015==
==02/07/2015==
 
Estimated g023 P159/170 PCR product concentration based on gel from yesterday. Probably around 7ng/ul. Annealed 2x20ul of each product, didn't add anything or purify, according to the SURVEYOR protocol. Cut with 2ul SURVEYOR nuclease and 1ul of enhancer. Used 3ul of elution solution for -nuc samples. Incubated at 42 for one minute, then it went back to room temp because I set the new PCR machine incorrectly. Noticed after about 10-15 minutes, incubated for another hour.
<br>
Added 1ul of cut stuff to 2ul stop solution and 17ul elution solution. Put at -20
<br>Didn't add stop solution to the rest, just directly analyzed on 1.5% agarose gel. Did some reading on agarose gels as well:
:voltage should be 4-10V/cm according to the distance between the electrodes. Our small gel box distance is 16cm so I will run at 5cm/V, or 80V.
:gels that are too thick will blur the bands. The thinnest gel comb we have is 0.1cmx0.4cm. I will have about 23ul of sample-1ul for bioanalyzer+4.4ul 6x loading dye or 0.0264cm^3. To determine the height of the gel I should use to fit all my sample in, I divide by the area of the comb.
::0.0264/0.4/0.1= 0.66cm
:to determine the volume of gel that corresponds to that gel height, i use the length and width of the gel cast tray: 11cmx9cm
::11cm*9cm*0.66cm=~65mL
:Cleaned the casting tray and the combs.
:I just poured a 60mL gel because I did the calculations wrong and then added 5mL of TAE while it was cooling. Used 6.5ul SYBR Safe
:A 1.5% gel should be good for resolving the smaller bands
:Will run for 45 minutes
<br><br>




Revision as of 17:32, 7 February 2015

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02/07/2015

Estimated g023 P159/170 PCR product concentration based on gel from yesterday. Probably around 7ng/ul. Annealed 2x20ul of each product, didn't add anything or purify, according to the SURVEYOR protocol. Cut with 2ul SURVEYOR nuclease and 1ul of enhancer. Used 3ul of elution solution for -nuc samples. Incubated at 42 for one minute, then it went back to room temp because I set the new PCR machine incorrectly. Noticed after about 10-15 minutes, incubated for another hour.
Added 1ul of cut stuff to 2ul stop solution and 17ul elution solution. Put at -20
Didn't add stop solution to the rest, just directly analyzed on 1.5% agarose gel. Did some reading on agarose gels as well:

voltage should be 4-10V/cm according to the distance between the electrodes. Our small gel box distance is 16cm so I will run at 5cm/V, or 80V.
gels that are too thick will blur the bands. The thinnest gel comb we have is 0.1cmx0.4cm. I will have about 23ul of sample-1ul for bioanalyzer+4.4ul 6x loading dye or 0.0264cm^3. To determine the height of the gel I should use to fit all my sample in, I divide by the area of the comb.
0.0264/0.4/0.1= 0.66cm
to determine the volume of gel that corresponds to that gel height, i use the length and width of the gel cast tray: 11cmx9cm
11cm*9cm*0.66cm=~65mL
Cleaned the casting tray and the combs.
I just poured a 60mL gel because I did the calculations wrong and then added 5mL of TAE while it was cooling. Used 6.5ul SYBR Safe
A 1.5% gel should be good for resolving the smaller bands
Will run for 45 minutes





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