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Sample Requirements

 
 

Fragment Length Analysis

 

How to Prepare Your Samples for Fragment Length Analysis

Volume:
min. 10 µl per sample arrayed in a 96-well plate.
 
 
The following protocol can be used to prepare the Ready-to-Load plate:
  • Dilute your size standard 1:100 with Hi-Di formamide
  • Distribute 10 µl size standard/formamide mix in an ABI3730 compatible 96 well plate (4titude catalog nr. 4ti-0730/C-SBC)
  • Dilute your PCR product e.g. 1:2-1:100 to reach the desired signal intensity
  • Add 1.2 µl diluted PCR product to 10 µl size standard/formamide mix
  • Empty wells must be filled with distilled water.
Ready-to-Load ABI3730XL 96 well plates:
FrameStar 96, with Upstand 4ti-0730/C-SBC
 
 
When you are sending labeled PCR products and Microsynth is adding the size standard you can choose from the following:
GeneScan 120 LIZ
GeneScan 350 ROX
GeneScan 400 HD ROX
GeneScan 500 LIZ
GeneScan 500 ROX
GeneScan 600 LIZ
ILS 600 CXR
MapMarker 1000 ROX
GeneScan 1200 LIZ
 
For any other size marker please contact us.
 
 
Filter Sets
Dye Set Name Matrix Type Blue Green Yellow Red Orange
Powerplex_4C 4 dye FAM JOE TAMRA CXR  
Powerplex_4C-RCT 4 dye FAM JOE TAMRA CXR  
DS-30_D 4 dye 6-FAM HEX NED ROX  
DS-30_D-RCT 4 dye 6-FAM HEX NED ROX  
DS-02_E5 5 dye dR110 dR6G dTAMRA dROX LIZ
DS-02_E5-RCT 5 dye dR110 dR6G dTAMRA dROX LIZ
DS-33_G5 5 dye 6-FAM VIC NED PET LIZ
DS-33_G5-RCT 5 dye 6-FAM VIC NED PET LIZ
Mic-G5 5 dye FAM ATTO532 ATTO550 ATTO565 Dyomics630
Mic-G5-RCT 5 dye FAM ATTO532 ATTO550 ATTO565 Dyomics630
 
RCT: This dye set reduces signal intensity and potential crosstalk of capillaries. Higher concentration peaks can be used without going offscale.
 
 
Filter set, Presence and type of the size standard should be declared while ordering and is customer responsibility.
 

 

Cell Line Authentication



Cell Pellets
Collect 1 to 5 Mio cells and wash the cell pellet twice in PBS or another appropriate buffer. Resuspend cell pellet in 0.5 ml of 70-90% ethanol and transfer to a 1.5 ml screw cab tube.

Isolated DNA
Please provide ≥50 µl of 50 ng/µl gDNA in Tris or low-EDTA buffer (10 mM Tris, 0.1 mM EDTA).



Mycoplasma Testing



Cultivate cells for a minimum of three days (72 h) without modifying the medium before collecting the supernatant for mycoplasma testing. Allow 80 - 90% confluence, but no higher, to avoid potential inhibition.


Sampling:

  • From adherent cell culture: collect supernatant directly from the culture flask.
  • From suspension culture: collect cells at the bottom of the tube before removing the supernatant. Ensure supernatant is free of cells by centrifugation of a subsample.
  • Collect 1000 μl of the cell-free supernatant in a screw cap tube or similar container that provides a tight seal during heat inactivation.
  • Centrifuge the sample at 750 x g for 2 minutes to pellet remaining cells and cellular debris.
  • Transfer 150 µl of the supernatant to a new 1.5 ml flip-cap tube.
  • Heat Inactivation: Incubate the tube containing the supernatant at 95°C for 10 minutes.
  • Label the tube with a Microsynth Mycoplasma barcode label. The label can be obtained in the webshop.