RNA Fragmentation – Rick’s trout RBC samples prepped earlier today (see below)

Samples were fragmented according to the Whole Transcriptome Kit protocol. Samples were then cleaned up using Invitrogen’s Modified RiboMinus Concentration Module, according to protocol. Briefly:

  • Added 1X volume of binding buffer (100uL)
  • Added equal volume of 100% EtOH so that [EtOH] = 50% (200uL)

Followed remainder of protocol and eluted with 20uL of H2O. Samples were spec’d.

Results:

Assuming the NanoDrop readings are accurate (according to the Whole Transcriptome Kit, these may NOT be accurate), got yields of ~93ng for the RBC Control sample and ~132ng for the RBC poly1:C sample. Transferred 1.5uL of each sample to separate 0.5mL tubes for submission to the Bioanalyzer. These were stored @ -80C in the “Bioanalyzer Samples” box. The remainder of the samples were stored @ -80C in the “Samples from Other Researchers” box.

mRNA Isolation – Rick’s trout RBC samples previously treated with Ribominus Kit (by Mac)

Was given ~0.5ug of each of these two RNA samples and processed them with Ambion’s microPolyA Purist Kit according to protocol. After elution, the samples were EtOH precipitated @ -80C for 30mins, pelleted 30mins 16,000g for 30mins, 4C. Supe removed, RNA washed with 1mL 70% EtOH and spun 10mins 16,000g, 4C. Supe removed. Resusupended in 8uL of The RNA Storage Solution and spec’d.

Results:

Yield of ~320ng for RBC Control sample and ~360ng for RBC poly1:C sample. Will proceed to Whole Transctiptome Kit fragmentation step.

mRNA Isolation – Gigas BB and DH samples previously treated with Ribominus Kit (by Mac)

Was given ~0.5ug of each of these two RNA samples and processed them with Ambion’s microPolyA Purist Kit according to protocol. After elution, the samples were EtOH precipitated @ -80C for 30mins, pelleted 30mins 16,000g for 30mins, 4C. Supe removed, RNA washed with 1mL 70% EtOH and spun 10mins 16,000g, 4C. Supe removed. Resusupended in 10uL of The RNA Storage Solution and gave back to Mac.

mRNA Isolation – Gigas BB and DH samples previously treated with Ribominus Kit (by Mac)

Was given 1ug of each of these two RNA samples and processed them with Promega’s PolyA Tract Kit according to protocol. After elution, the samples were EtOH precipitated @ -20C for 30mins, pelleted 30mins 16,000g for 30mins, 4C. Supe removed, RNA washed with 1mL 70% EtOH and spun 15mins 16,000g, 4C. Supe removed. Resusupended in 15uL of 0.1% DEPC-H2O and spec’d.

Results: No measurable amount of RNA in either sample. Samples were stored @ -80C.

HRMs – Lake Trout SNPs (HRM_white-05 & HRM_white_06)

HRM_white-05

The following primers from primer plate LTP01 will be used for analysis of Rick’s Lake Trout DNA1 plate (from 4/28/2009): G10, C11, H11, A12. HRM set up is here. It is the same as that used on 20090903. A 1:10 dilution plate (from 20090903) of Rick’s Lake Trout DNA1 plate (from 4/28/2009) was used. This means approximately 20ng of DNA used in each rxn. The robot was used to add 1uL of DNA from the 96-well plate to the appropriate wells of the 384-well HRM rxn plate. Plate was spun to collect the DNA at the bottom of the wells. The wells were visually inspected to ensure that each received the DNA. 1uL of DNA was manually added to those wells that did not receive sample.

The master mix for each primer set was then manually dispensed to the appropriate wells in the 384-well HRM rxn plate using a “matrix” auto-pipette. The real-time PCR was run in a Roche LightCycler480 machine with the following cycling parameters:

95C – 15mins

45 cycles of:

95C – 10s

60C – 15s

72C – 25s

After the completion of the real-time run, the plate was put through the High Melt Curve protocol.

 

 

HRM_white-06

The following primers from primer plate LTP01 will be used for analysis of Rick’s Lake Trout DNA1 plate (from 4/28/2009): E12. HRM set up is here. It is the same as that used on 20090903. A 1:10 dilution plate (from 20090903) of Rick’s Lake Trout DNA1 plate (from 4/28/2009) was used. This means approximately 20ng of DNA used in each rxn. The robot was used to add 1uL of DNA from the 96-well plate to the appropriate wells of the 384-well HRM rxn plate. Plate was spun to collect the DNA at the bottom of the wells. The wells were visually inspected to ensure that each received the DNA. 1uL of DNA was manually added to those wells that did not receive sample.

The master mix for each primer set was then manually dispensed to the appropriate wells in the 384-well HRM rxn plate using a “matrix” auto-pipette. The real-time PCR was run in a Roche LightCycler480 machine with the following cycling paramters:

95C – 15mins

45 cycles of:

95C – 10s

60C – 15s

72C – 25s

After the completion of the real-time run, the plate was put through the High Melt Curve protocol.

HRMs – Lake Trout SNPs (HRM_white-03 & HRM_white-04)

HRM_white-03

The following primers from primer plate LTP01 will be used for analysis of Rick’s Lake Trout DNA1 plate (from 4/28/2009): E4, B5, A7, D7. HRM set up is here. It is the same as that used on 20090903. A 1:10 dilution plate (from 20090903) of Rick’s Lake Trout DNA1 plate (from 4/28/2009) was used. This means approximately 20ng of DNA used in each rxn. The robot was used to add 1uL of DNA from the 96-well plate to the appropriate wells of the 384-well HRM rxn plate. Plate was spun to collect the DNA at the bottom of the wells. The wells were visually inspected to ensure that each received the DNA. 1uL of DNA was manually added to those wells that did not receive sample.

The master mix for each primer set was then manually dispensed to the appropriate wells in the 384-well HRM rxn plate using a “matrix” auto-pipette. The real-time PCR was run in a Roche LightCycler480 machine with the following cycling paramters:

95C – 15mins

45 cycles of:

95C – 10s

60C – 15s

72C – 25s

After the completion of the real-time run, the plate was put through the High Melt Curve protocol.

 

HRM_white-04

The following primers from primer plate LTP01 will be used for analysis of Rick’s Lake Trout DNA1 plate (from 4/28/2009): H7, B8, C8, F10. HRM set up is here. It is the same as that used on 20090903. A 1:10 dilution plate (from 20090903) of Rick’s Lake Trout DNA1 plate (from 4/28/2009) was used. This means approximately 20ng of DNA used in each rxn. The robot was used to add 1uL of DNA from the 96-well plate to the appropriate wells of the 384-well HRM rxn plate. Plate was spun to collect the DNA at the bottom of the wells. The wells were visually inspected to ensure that each received the DNA. 1uL of DNA was manually added to those wells that did not receive sample.

The master mix for each primer set was then manually dispensed to the appropriate wells in the 384-well HRM rxn plate using a “matrix” auto-pipette. The real-time PCR was run in a Roche LightCycler480 machine with the following cycling paramters:

95C – 15mins

45 cycles of:

95C – 10s

60C – 15s

72C – 25s

After the completion of the real-time run, the plate was put through the High Melt Curve protocol.

HRM – Lake Trout SNPs (HRM_white-02)

HRM_white-02

The following primers from primer plate LTP01 will be used for analysis of Rick’s Lake Trout DNA plate (from 4/28/2009): A3, D3, G3, A4. HRM set up is here. It is the same as that used on 20090903. A 1:10 dilution plate (from 20090903) of Rick’s Lake Trout DNA1 plate (from 4/28/2009) was used. This means approximately 20ng of DNA used in each rxn. The robot was used to add 1uL of DNA from the 96-well plate to the appropriate wells of the 384-well HRM rxn plate. Plate was spun to collect the DNA at the bottom of the wells. The wells were visually inspected to ensure that each received the DNA. 1uL of DNA was manually added to those wells that did not receive sample.

The master mix for each primer set was then manually dispensed to the appropriate wells in the 384-well HRM rxn plate using a “matrix” auto-pipette. The real-time PCR was run in a Roche LightCycler480 machine with the following cycling paramters:

95C – 15mins

45 cycles of:

95C – 10s

60C – 15s

72C – 25s

After the completion of the real-time run, the plate was put through the High Melt Curve protocol.

Results:

HRM – Lake Trout SNPs (HRM-white-01)

The following primers from primer plate LTP01 will be used for analysis of Rick’s Lake Trout DNA plate (from 4/28/2009): A1, C1, H1, B2. So, that’s 4 primer sets x 96 DNA samples = 384. HRM set up is here. A 1:10 dilution plate of Rick’s Lake Trout DNA1 plate (from 4/28/2009) was made for HRM. This means approximately 20ng of DNA used in each rxn. The robot was used to add 1uL of DNA from the 96-well plate to the appropriate wells of the 384-well HRM rxn plate. Plate was spun to collect the DNA at the bottom of the wells. The wells were visually inspected to ensure that each received the DNA. 1uL of DNA was manually added to those wells that did not receive sample (B23, C23).

The master mix for each primer set was then manually dispensed to the appropriate wells in the 384-well HRM rxn plate using a “matrix” auto-pipette. The real-time PCR was run in a Roche LightCycler480 machine with the following cycling paramters:

95C – 15mins

45 cycles of:

95C – 10s

60C – 15s

72C – 25s

After the completion of the real-time run, the plate was put through the High Melt Curve protocol.

Results:

qPCR – HRM Lake Trout SNP primer test

Tested out the plate of Lake Trout primers (LTP01 – forward and reverse combined) for SNP detection. qPCR was performed using Roche 2x HRM M.M. qPCR set up is here. Cycling params are as follows:

95C – 10mins

40 cycles of:

95C – 10s

60C – 15s

72C – 25s

Plate layout matches the primer plate layout.

Results: The following wells have signals and good, clean melting curves: A1, C1, H1, B2, A3, D2, G3, A4, E4, B5, A7, D7, H7, B8, C8, F10, G10, C11, H11, A12, E12. These are potential candidates for SNP analysis. Will test HRM analysis using these primers, each on a subset of Lake Trout DNA samples to see whether or not they’ll be truly useful for analyzing the full plate of DNA.

Primers – Lake Trout Primers for HRM

The two primer plates (LTP01F, LTP01R) were received today. Primers (supplied as 10 nmoles of each) will be reconstituted with 100uL of PCR H2O to make a Cf of 100uM. Forward and reverse primers will be combined in a new plate, in equal volumes, to give a Cf of 50uM. These combined primers will be used to test them on pooled lake trout DNA to identify functional primer pairs for use in HRM.