{"id":1336,"date":"2013-04-10T13:39:17","date_gmt":"2013-04-10T17:39:17","guid":{"rendered":"http:\/\/www.ambrosiasymbiosis.org\/labprotocols\/picogreen-on-the-real-time-pcr-machine\/"},"modified":"2016-01-05T10:59:06","modified_gmt":"2016-01-05T15:59:06","slug":"picogreen-on-the-real-time-pcr-machine","status":"publish","type":"post","link":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/picogreen-on-the-real-time-pcr-machine\/","title":{"rendered":"Picogreen on the Real TIme PCR Machine"},"content":{"rendered":"

Picogreen on the RT PCR Machine<\/b><\/p>\n

 <\/p>\n

Per Chris Dervinis, Sr Biologist and Forest Genomics Lab Manager, SFRC, Univ. of FL :<\/p>\n

“We use the sybr green filters for the assay, which is not mentioned in the protocol.\u00a0 One thing to keep in mind is that the linear range for picogreen in these smaller assay is lower than it is with a plate reader and larger volume size, so you will need to be sure that your samples are under 50ng\/ul or you will need to dilute for an accurate approximation.”<\/i><\/p>\n

Plate Setup<\/span><\/b><\/p>\n

1.Set up the standards using the Lambda DNA \u2013 Component C (100 ng\/\u00b5l) with a total volume of 5 \u00b5l<\/p>\n

For example:<\/b> Make a 10 ng\/\u00b5l Standard from the Lambda DNA<\/p>\n

Add TE to the plate, according to the scheme below:<\/p>\n

 <\/p>\n\n\n\n\n\n\n\n\n\n\n\n
<\/td>\n\n

1<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

7<\/p>\n<\/td>\n

\n

8<\/p>\n<\/td>\n

\n

9<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

11<\/p>\n<\/td>\n

\n

12<\/p>\n<\/td>\n<\/tr>\n

A<\/td>\n\n

0 \u00b5l<\/p>\n<\/td>\n

\n

0 \u00b5l<\/p>\n<\/td>\n

\n

4 \u00b5l<\/p>\n<\/td>\n<\/tr>\n

B<\/td>\n\n

1 \u00b5l<\/p>\n<\/td>\n

\n

1 \u00b5l<\/p>\n<\/td>\n<\/tr>\n

C<\/td>\n\n

2 \u00b5l<\/p>\n<\/td>\n

\n

2 \u00b5l<\/p>\n<\/td>\n<\/tr>\n

D<\/td>\n\n

2.5 \u00b5l<\/p>\n<\/td>\n

\n

2.5 \u00b5l<\/p>\n<\/td>\n<\/tr>\n

E<\/td>\n\n

3 \u00b5l<\/p>\n<\/td>\n

\n

3 \u00b5l<\/p>\n<\/td>\n<\/tr>\n

F<\/td>\n\n

3.5 \u00b5l<\/p>\n<\/td>\n

\n

3.5 \u00b5l<\/p>\n<\/td>\n<\/tr>\n

G<\/td>\n\n

4 \u00b5l<\/p>\n<\/td>\n

\n

4 \u00b5l<\/p>\n<\/td>\n<\/tr>\n

H<\/td>\n\n

5 \u00b5l<\/p>\n<\/td>\n

\n

5 \u00b5l<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Add DNA Standard (10 ng\/ \u03bcL \u2013 kit supplies DNA standard 100 ng\/ \u03bcL) and the samples, according to the scheme bellow:<\/p>\n

 <\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
<\/td>\n\n

1<\/p>\n<\/td>\n

\n

2<\/p>\n<\/td>\n

\n

3<\/p>\n<\/td>\n

\n

4<\/p>\n<\/td>\n

\n

5<\/p>\n<\/td>\n

\n

6<\/p>\n<\/td>\n

\n

7<\/p>\n<\/td>\n

\n

8<\/p>\n<\/td>\n

\n

9<\/p>\n<\/td>\n

\n

10<\/p>\n<\/td>\n

\n

11<\/p>\n<\/td>\n

\n

12<\/p>\n<\/td>\n<\/tr>\n

A<\/td>\n\n

5 \u00b5l (50ng)<\/p>\n<\/td>\n

\n

5 \u00b5l<\/p>\n<\/td>\n

\n

1 \u03bcL<\/p>\n<\/td>\n

<\/td>\n<\/tr>\n
B<\/td>\n\n

4 \u00b5l (40 ng)<\/p>\n<\/td>\n

\n

4 \u00b5l<\/p>\n<\/td>\n

<\/td>\n<\/tr>\n
C<\/td>\n\n

3 \u00b5l (30 ng)<\/p>\n<\/td>\n

\n

3 \u00b5l<\/p>\n<\/td>\n

<\/td>\n<\/tr>\n
D<\/td>\n\n

2.5 \u00b5l (25 ng)<\/p>\n<\/td>\n

\n

2.5 \u00b5l<\/p>\n<\/td>\n

<\/td>\n<\/tr>\n
E<\/td>\n\n

2 \u00b5l (20 ng)<\/p>\n<\/td>\n

\n

2 \u00b5l<\/p>\n<\/td>\n

<\/td>\n<\/tr>\n
F<\/td>\n\n

1.5 \u00b5l (15 ng)<\/p>\n<\/td>\n

\n

1.5 \u00b5l<\/p>\n<\/td>\n

<\/td>\n<\/tr>\n
G<\/td>\n\n

1 \u00b5l (10 ng)<\/p>\n<\/td>\n

\n

1 \u00b5l<\/p>\n<\/td>\n

<\/td>\n<\/tr>\n
H<\/td>\n\n

0 \u00b5l (0 ng)<\/p>\n<\/td>\n

\n

0 \u00b5l<\/p>\n<\/td>\n

<\/td>\n<\/tr>\n
<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

1.Add 25 \u00b5l of Picogreen solution into each well<\/p>\n

    \n
  1. Picogreen dilution: (1 Component A: 200 TE)<\/li>\n<\/ol>\n

    NOTE: Protect Picogreen solution from light (photo-sensitive)<\/p>\n

    1.Add the PCR flat caps over each column with the Strip Cap Tool (avoid touching the top of caps with your hands because the reading must pass through the caps)<\/p>\n

      \n
    1. Vortex and spin down<\/li>\n<\/ol>\n

      RT PCR Set up<\/span><\/b><\/p>\n

      Lamp Warm up takes 20 mins so have the lamp warming up before starting plate setup.<\/p>\n

      1.Open the realplex program\u00a0(password is “e”). If the program is not recognizing the thermocycler, make sure the USB \u00a0connection to the computer is in the right port (both the port and the cord are marked with marker). All other connections can be checked based on the arrangement in the manual (on computer desktop).<\/p>\n

        \n
      1. Choose \u201cQuantitative Plate Read\u201d<\/li>\n
      2. Set the plate grid\n
          \n
        1. Highlight wells that are for standard and click on \u201cStandard\u201d (right hand side)<\/li>\n
        2. Highlight wells that are for samples and click on \u201cUnknown\u201d (right hand side)<\/li>\n
        3. Highlight ALL <\/span><\/b>wells that will be read and click SYBR<\/li>\n
        4. Set the values for the Standards (for example: 50, 40, 30, 25, 20, 15, 10, 0)\n
            \n
          1. Set the 2 standard as replicates (right hand side) so the 2 readings will be averaged for 1 point on the standard curve<\/li>\n
          2. Change the quantity from \u201ccopies\u201d to \u201cnanogram\u201d (right hand side)\n
              \n
            1. It will ask for the conversion; Click \u201cDo Not Convert\u201d<\/li>\n
            2. Click Run<\/li>\n
            3. Click Pre-Read<\/li>\n
            4. To analyze the data, click \u201cAnalysis\u201d on the top right hand side, click \u201cResults\u201d tab to view Standard curve, Text Report, etc.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n

              Save and export data to powerpoint or excel<\/p>\n","protected":false},"excerpt":{"rendered":"

              Picogreen on the RT PCR Machine   Per Chris Dervinis, Sr Biologist and Forest Genomics Lab Manager, SFRC, Univ. of FL : “We use the sybr green filters for the assay, which is not mentioned in the protocol.\u00a0 One thing to keep in mind is that the linear range for picogreen in these smaller assay […]<\/p>\n","protected":false},"author":7,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_s2mail":"yes","footnotes":""},"categories":[3],"tags":[],"class_list":["post-1336","post","type-post","status-publish","format-standard","hentry","category-general-molecular-work"],"_links":{"self":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1336","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/users\/7"}],"replies":[{"embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/comments?post=1336"}],"version-history":[{"count":4,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1336\/revisions"}],"predecessor-version":[{"id":1879,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/posts\/1336\/revisions\/1879"}],"wp:attachment":[{"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/media?parent=1336"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/categories?post=1336"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.ambrosiasymbiosis.org\/labprotocols\/wp-json\/wp\/v2\/tags?post=1336"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}