QPCR - Lin Lab - Revision history

Davebrid: /* Before the run: */ modified machine setup

2009-05-28T12:13:48Z

Before the run:: modified machine setup

← Older revision		Revision as of 12:13, 28 May 2009
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	==Before the run:==		==Before the run:==
	#Click on ~~“Shortcut to Sybr plate”~~ icon.		#Spin down plate 1 min at 3000 RPM in an empty 96 well plate using the Lin lab centrifuge
	#Click on ~~“Instrument” tab and change volume of rxn. to 20uL~~. With new primers, may want to run the dissociation step to see if one peak (or more) is generated by the primers. This step adds about an extra hour to the run time. Can eliminate this step for primers which have already been analyzed in this way.		#Press power button.
	#Go to File menu and then Save As. Go up one folder to ABI folder then to the Results folder, then save in the Saltiel folder ~~(in my folder).~~		#Open tray and put plate into tray.
	~~#Close~~ the ~~program (Shortcut to Sybr plate window).~~		#Click on “7300 System Software” icon.
	~~#Reopen the program through the 7300 System Software icon. Open the new saved file and~~ the ~~7300 system should then initialize~~.		#Click on open then in the directory Templates select SYBR Plate or SYBR Plate (no dissociation). With new primers, may want to run the dissociation step to see if one peak (or more) is generated by the primers. This step adds about an extra hour to the run time. Can eliminate this step for primers which have already been analyzed in this way.
	#Under the Instrument tab, hit “Start.” ~~Note:~~ It takes a few minutes for the run to begin after “Start” is hit. Sometimes the run time is incorrectly calculated by the software.		#Click on “Instrument” tab and check that reaction volume is 20uL.
			#Go to File menu and then Save As. Go up one folder to ABI folder then to the Results folder, then save in the Saltiel folder with the date as the filename.
			#Under the Instrument tab, hit “Start.”
			#Wait until a estimated run time is shown. It takes a few minutes for the run to begin after “Start” is hit. Sometimes the run time is incorrectly calculated by the software.

	==After the run:==		==After the run:==

Davebrid: /* PCR Program: */

2009-05-27T22:02:54Z

PCR Program:

← Older revision		Revision as of 22:02, 27 May 2009
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	{\| border = "1"		{\| border = "1"
	\|Stage 1 \|\| Stage 2 \|\| Stage 3 \| ~~colspan = "4"~~\| Dissociation		\|Stage 1 \|\| Stage 2 \|\| Stage 3 \|\| \|\| Dissociation
	\|-		\|-
	\| Rep 1 \|\| Rep 1 \|\| Rep 40 \| ~~colspan = "4"~~\| Stage 4		\| Rep 1 \|\| Rep 1 \|\| Rep 40 \|\| \|\| Stage 4
	\|-		\|-
	\| \|\| \|\| \| ~~colspan = "4"~~\| Rep 1		\| \|\| \|\| \|\| \|\| Rep 1
	\|-		\|-
	\| 50C \|\| 95C \|\| 95C \|\| 60C \|\| 95C \|\| 60C \|\| 95C		\| 50C \|\| 95C \|\| 95C \|\| 60C \|\| 95C \|\| 60C \|\| 95C

Davebrid: /* PCR Program: */

2009-05-27T21:58:17Z

PCR Program:

← Older revision		Revision as of 21:58, 27 May 2009
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	{\| border = "1"		{\| border = "1"
	\|Stage 1 \|\| Stage 2 \|\| Stage 3 \|\| Dissociation		\|Stage 1 \|\| Stage 2 \|\| Stage 3 \| colspan = "4"\| Dissociation
	\|-		\|-
	\| Rep 1 \|\| Rep 1 \|\| Rep 40 \|\| Stage 4		\| Rep 1 \|\| Rep 1 \|\| Rep 40 \| colspan = "4"\| Stage 4
	\|-		\|-
	\| \|\| \|\| \|\| Rep 1		\| \|\| \|\| \| colspan = "4"\| Rep 1
	\|-		\|-
	\| 50C \|\| 95C \|\| 95C \|\| 60C \|\| 95C \|\| 60C \|\| 95C		\| 50C \|\| 95C \|\| 95C \|\| 60C \|\| 95C \|\| 60C \|\| 95C

Davebrid: /* PCR Program: */

2009-05-27T21:55:30Z

PCR Program:

← Older revision		Revision as of 21:55, 27 May 2009
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	==PCR Program:==		==PCR Program:==

	{\|		{\| border = "1"
	\|Stage 1 \|\| Stage 2 \|\| Stage 3 \|\| Dissociation		\|Stage 1 \|\| Stage 2 \|\| Stage 3 \|\| Dissociation
	\|-		\|-

Davebrid: /* After the run: */

2009-05-27T21:55:09Z

After the run:

← Older revision		Revision as of 21:55, 27 May 2009
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	PCR Program:		==PCR Program:==

	Stage 1		{\|
	Stage 2		\|Stage 1 \|\| Stage 2 \|\| Stage 3 \|\| Dissociation
	Stage 3		\|-
			\| Rep 1 \|\| Rep 1 \|\| Rep 40 \|\| Stage 4
	Dissociation		\|-
			\| \|\| \|\| \|\| Rep 1
			\|-
	Rep 1		\| 50C \|\| 95C \|\| 95C \|\| 60C \|\| 95C \|\| 60C \|\| 95C
	Rep 1		\|-
	Rep 40		\|2 min \|\| 10 min \|\| 15 sec \|\| 1 min \|\| 15 sec\|\| 30 sec\|\| 15 sec
			\|}
	Stage 4






	Rep 1


	50C
	95C
	95C
	60C
	95C
	60C
	~~95C~~
	2 min
	10 min
	15 sec
	1 min
	15 sec
	30 sec
	15 sec

	Protocol written for 96-well Real-time PCR machine in Jiandie Lin lab on 10/4/2006.		Protocol written for 96-well Real-time PCR machine in Jiandie Lin lab on 10/4/2006.

Davebrid: Created page with '==Before the run:== #Click on “Shortcut to Sybr plate” icon. #Click on “Instrument” tab and change volume of rxn. to 20uL. With new primers, may want to run the dissociat...'

2009-05-27T21:51:18Z

Created page with '==Before the run:== #Click on “Shortcut to Sybr plate” icon. #Click on “Instrument” tab and change volume of rxn. to 20uL. With new primers, may want to run the dissociat...'

New page

==Before the run:==
#Click on “Shortcut to Sybr plate” icon.
#Click on “Instrument” tab and change volume of rxn. to 20uL. With new primers, may want to run the dissociation step to see if one peak (or more) is generated by the primers. This step adds about an extra hour to the run time. Can eliminate this step for primers which have already been analyzed in this way.
#Go to File menu and then Save As. Go up one folder to ABI folder then to the Results folder, then save in the Saltiel folder (in my folder).
#Close the program (Shortcut to Sybr plate window).
#Reopen the program through the 7300 System Software icon. Open the new saved file and the 7300 system should then initialize.
#Under the Instrument tab, hit “Start.” Note: It takes a few minutes for the run to begin after “Start” is hit. Sometimes the run time is incorrectly calculated by the software.

==After the run:==

(Steps 1 and 2 are only necessary if the file is not examined immediately after the run.)

#Click on the “Shortcut to Sybr plate” icon.
#Go to File menu then Open. Go up one folder to the Results folder and open the Saltiel folder. Then open my folder to get to the file to open.
#Hit the “Results” tab then the “Amplification” plot to review the amplication curves. Highlight cells on the plate grid to see the curves associated with the cells.
#Check the Dissociation Curve to see if there is one peak for particular primer pairs. Again, highlight cells on the plate grid to see the curves associated with the cells.
#Go to “Amplification Plot” or go to the Analysis menu. Select Auto CT then select Analyze (also under the Analysis menu). Can also set the threshold manually. Don’t worry about the baseline as long as the end cycle isn’t set at too high a cycle number and might interfere with the analysis. The scale on the Y-axis can be adjusted on the graph to make it easier to select the threshold. Click on the Y-axis to open a pop-up window to manually set the Y-axis.
#Hit “Report” and highlight the cells used. Go to “Export” then “Results” under the File menu. There will be a .csv file which Excel can read.
#Export the report in my folder inside the “Results” folder. Exit the software and save changes to “Absolute Quantification.”
#To transfer the USB drive, insert the device then go to My Computer to open the USB window (i.e. the removeable disk window) if it does not open automatically. Then go to “Shortcut to results”icon to open that window. Drag and drop the .sds (raw data) and .csv (Excel spreadsheet) files into the USB window. Right click on the green arrow hardware button at the bottom right of the screen. Then hit the U3 icon next to the green arrow to open the U3 window. Hit the Eject button to safely remove the USB drive.

PCR Program:

Stage 1
Stage 2
Stage 3

Dissociation

Rep 1
Rep 1
Rep 40

Stage 4

Rep 1

50C
95C
95C
60C
95C
60C
95C
2 min
10 min
15 sec
1 min
15 sec
30 sec
15 sec

Protocol written for 96-well Real-time PCR machine in Jiandie Lin lab on 10/4/2006.