Question: Can I use custom panels with the SPRIselect Library Concentration Protocol in the Targeted Gene Expression assay?
Answer: The Demonstrated Protocol for SPRIselect Library Concentration is compatible with both fully custom and add-on panels. There are a few protocol changes required if using a custom or add-on panel compared to a pre-designed panel and these changes are mentioned in the Demonstrated Protocol.
The modifications are also listed below:
1) Fully custom panels
Follow the panel dilution guidelines in the Targeted Gene Expression User Guide to dilute the fully custom panel to an appropriate working dilution before use.
Follow the Demonstrated Protocol for SPRIselect Library Concentration, with the following protocol modifications:
At step 2.a., prepare the Modified Hybridization Master Mix according to this table:
|
|
1x (µl) |
|
Universal Blockers |
2.0 |
|
Hyb Buffer |
9.5 |
|
Hyb Enhancer |
3.0 |
|
Fully Custom Panel Working Dilution |
4.5 |
|
Total |
19 |
2) Add-on panels
Follow the panel dilution guidelines in the Targeted Gene Expression User Guide to dilute the add-on panel to an appropriate working dilution before use.
Follow the Demonstrated Protocol for SPRIselect Library Concentration, with the following protocol modifications:
At step 2.a., prepare the Modified Hybridization Master Mix according to this table:
|
|
1x (µl) |
|
Universal Blockers |
2.0 |
|
Hyb Buffer |
9.5 |
|
Hyb Enhancer |
3.0 |
|
Add-on Panel Working Dilution |
2.2 |
|
Pre-designed Panel |
4.5 |
|
Total |
21.2 |
At step 2.k, add 21.2 µl of Modified Hybridization Master Mix.
At step 2.n, transfer 17 µl of supernatant into a new tube and proceed with Library Hybridization.
It may be beneficial to add an extra PCR cycle to the post-capture library amplification step to account for the slight loss in the material due to not carrying forward all of the supernatant at step 2.n