Cutting visible stained gel with the use of external pick list.

Transcription

1 Cutting visible stained gel with the use of external pick list. Summary: 1) Image gel acquisition on the Xcise imager. 2) Set the Anchor points on the Xcise gel image. 3) Up load the external image. 4) Up load and applied the tds (pick list) file on the external image. 5) Set the anchor points on the external image in the exact same order as set in the internal Xcise gel image. 6) Set the wet position points on the external image. 7) Fuse the two images. 8) Check the position of the xcise points on the fused image. 9) Cut sample gel as usual procedure. - To cut the fluorescence gel on the Xcise, obtain the gel image on the external fluorescence imager first, and then transfer its 8 bit tiff image file (Only 8bit image is supported) and sample gel to the Xcise. - To cut spots by using a pick list, you have to make the tds export file first and then transfer it to the Xcise. Xcise use the Progenesis analysis results file format for pick list: to generate such.tds file format, see tds format page. To get the pick list file converter (Decyder format to.tds format), contact: granjeau@tagc.univ-mrs.fr - 1 -

2 Internal gel Process 1- Image acquisition. Place the stained gel on the Xcise gel frame and set the cover frame. Transfer the sample gel to the Xcise scanner. Don t forget to wet it. Set the Xcise scanner lid cover for large gel scanning. 1-1) Open the GelScan tab. 1-2) Enter Proj., Exp., Name (image name) and Barcode (2nd comment) 1-3) Click on the Scan button and then select the appropriate gel format Mini-Gel to DB or large gel to DB. A message windows come out. Click on [Yes] to start scanning

3 2- Image process. 2-1) Open the GelAnalysis tab. 2-2) Select the internal image in the navigation window, and click on the [Load] button

4 2-3) Click on the [View] button and select the [Anchor Mode]. 2-4) To set the Anchor points, click [Ctrl] and the left mouse button on the markers you select

5 2-5) Click on the Files on the menu bar and select Save Point File to DB. 2-6) Click on the Files on the menu bar and select Exit

6 External gel image process 3- Uploading external images. Transfer the gel image file and the tds file on the Images folder and spot_files folder to the win_pub directory on the Xcise through LAN. 3-1) Click on the [Upload Image] button in the Xcise main window. 3-2) Enter Image_Name and Barcode to save the image in the Data Base, and then click on 3-3) Select the external fluorescence image file in the win_pub directory

7 3-4) Click on [OK] to load the file. 3-5) Click on [OK] and click on [Dismiss]. The image is loading in the navigation window

8 4- Image process. 4-1) Select the GelAnalysis tab. 4-2) Select the external image in the navigation window, and click on the [Load] button. 4-3) Click on the [View] button and select the [All Points]

9 4-4) Set the cutting spots according to the external pick list. Click on Files and Load Point File. 4-5) Select the tds file in the winpub directory. Click on [OK] to load the file. You will automatically find the cutting spots on the image. Check the spot position

10 4-6) Click on Point on the menu bar and [Select All Points]. 4-7) Click on Point and select [Set Selected Pt Type] and also select [Xcise] mode. Points defined tds in All Mode are copied into the Xcise Mode

11 4-8) Click on Files and select Save Point File to DB. 4-9) Change from the [All] to the [Anchor Mode] mode. 4-10) Set the anchor points in the exact same order as set in the Xcise internal gel image. To set the Anchor points, click [Ctrl] and the left mouse button on the same markers you select

12 4-11) Click on the Files and select Save Point File to DB. 4-12) Change from the [Anchor Mode] to the [Wet Pos] mode. 4-13) Set the wet position points as wondered on the cutting method (4 spots needed). To set the wet position points, click [Ctrl] and the left mouse button on the markers you select

13 4-14) Click on the Files and select Save Point File to DB. 4-15) Click on the Files and Exit

14 5- Image matching 5-1) Select the GelAnalysis tab. 5-2) Hold [Ctrl] key and select both the external image and the Xcise image with the left mouse button. 5-3) Click on the [Load] button to load two images. The two images had to be highlighting on the loading window. If not, hold [Ctrl] key and select both the external image and the Xcise image with the left mouse button

15 5-4) Click on the [Register] button. This new Register image in database window appears: 5-5) Select the Xcise image (the one you performed with the Xcise scanner) for the Reference Image in the Register image in database window. Click on [OK], wait few seconds

16 5-6) You have a new image file with w in the navigation window. 5-7) Select the ****_w image in the navigation window

17 5-8) Click on the [Load] button and the [View] button and select the [Xcise Mode]. 5-9) Check the cutting spots on the Xcise fluorescence merged gel image. If any changes are performed, click on the [files] button and then click on [save point file to DB] 5-10) Click on the Files and Exit

18 5-11) Select the internal Xcise image in the navigation window, and click on the [Load] button and the [View] button to select the [Xcise Mode]. 5-12) Click on the [Files] button and the [Load point file from DB] button and select the Entry_id: Select the one from the ****_w image in the navigation window. Click on OK. Overlaid image of the theoretical cut spot positions will be displayed

19 5-13) Check the cutting spots coordinates on the Xcise gel image. If any changes are performed, click on the [files] button and then click on [save point file to DB] 5-14) Click on the Files and Exit

20 6- Spot cutting 6-1) Go to the Liquid Delivery tab. Check the MTP s state. 6-2) Go to the Gel Cutting tab

21 6-3) Click on the [Load gel from DB]. Message windows come out. Click twice on [Yes] to load the w merge image. Check the gel information: number of spots ; the image name 6-4) Check the set plate ID

22 6-5) Click on the [gel cutting]. Cut sample gel as usual procedure: Select [auto], click the type and position of gel frame