Submitted 1.5 plates for Sanger sequencing. Dungan isolates prepared by me, Lake Trout HRM prepared by Rony and Emma’s differential display cloning samples prepared by her. All primers were prepped by me. See the sequencing log for samples and plate layout.
Tag Archives: high resolution melt curve
Sequencing – Lake Trout HRM
This is a second submission of 12 individuals from 8 primer sets. The previous sequencing run was botched because I used the combined primer plate instead of a single (forward or reverse) primer for submission.
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:
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.
qPCR – Carita Primer Test for High Resolution Melt (HRM) Curve Analysis
Ran a qPCR on Rick’s Lake Trout DNA from 4/28/2009 using primers in Carita’s CMA01 Primer Plate (Excel file). DNA was pooled (2uL from each sample), spec’d and diluted to 10ng/uL. qPCR set up is here. Plate layout matches Carita’s primer plate layout. Since we’re just looking for positive/negative samples, I ran this on the Opticon 2 despite the recent “problems” we’ve been having with it. Cycling params used with the 2x HRM M.M. are as follows:
95C – 10mins
40 cycles of:
95C – 10s
60C – 15s
72C – 25s
Results: Overall, looks good. Negative control is clean. Based on signal strength and clean, tight melting curves, the following primer sets (location in CMA01 primer/qPCR plates) will be used for HRM analysis tomorrow: C11, D3, E12, D3. Actually, never mind. Steven’s sent me contig info for the lake trout so we’ll just order and test primers that are more likely to produce good data instead of worrying about the salmonid primers.