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This page last changed on Jun 15, 2011 by preston.
This email discusses the changes that have to occur to have HAB arrays and qPCR from one sample event. It also incorporates the other possible code changes that have to occur on NEO. We should also keep in mind running the DA arrays in concert and how this affects the plumbing and valve moves of the PRV.
1. Copy NEO's current Flash to Tornado
6/14/11 Tornado/ESP/NEOtombstoneCode11jun14
2. Update code on NEO (Brent has already updated ruby code) and determine what is now "broken"
6/14/11 moved to bufflehead. Use user preston. Problems with /var/log setup and the correct path for mounting bufflehead. If it loose home or fails use /sbin/service nfsmount start to regain connection. You must be a superuser. Brent will have to look at this when he returns.
3. Rig with Dummy MFB to make seeing core moves easier
4. Determine if the AIV lines (to PRV and CBV) need to be changed.
a. To match other instruments and handle the larger lysate volume generated by the HAB protocol
6/15/11 With the current lines on NEO and new code only recover 700ul of lysate. Prime is not correct we loose too much. After CS.push to 0 the line between the PRV and PTV is completely filled with lysate which is all sent to waste.
5. Do we have enough lysate volume for delivery of 1ml lysate to the array and up to 250 ul for the qPCR?
a. update PRVprocess.rb to handing the priming of the larger volume (depending on length of CBV-AIV-PRV line this may effect the positioning of the lysate to the PRV for the bacSHAmfb protocol)
6. Update code to deliver 1ml lysate (undiluted ) to array. Current protocol dilutes to 1.5M GusCN for BAC array.
7. Create and testing of habSHAmfb protocol based on the structure of the bacSHAmfb
a. Start with "normal protocol" 250ul of lysate and 225 ul of SPEdiluent
8. Testing of dilution of Lysate protocol
6/15/11 Dilution protocol will have to include a rinse of the mixloop. The transferPRV protocol uses the mixloop to depressurize the lines. Lots of concentrated lysate.
9. Timing of array hybridization with lysate dilution step --incorporation into habSHAmfb and mfb protocol
-----update of bacSHAmfb is not necessary for Pier deployment
10. CC clamp has to get the heavier springs
The second email Diluting Lysate for qPCR on ESP (Email 2 of 2)-- is exactly for that lysate dilution by the core prior to delivery to the MFB.
Brent added code to pass in an optional block in the loadMFBslug protocol, which will make its implementation much easier. It will have to be determined if the timing of diluting the lysate fits into the current time alloted for the hybridization of the array (if one is being run on the same sample.) This code starts once the concentrated lysate has been primed to the PRV.
The Major steps are:
0.5 rinse of the mixloop (Add to beginning of dilution protocol before loadMFBslug starts)
1. Airs out mixloop line (Currently done in loadMFBslug)
2. Dilution of Lysate (start with 1:10 dilution). Pulls lysate 0.1 ml then GuSCN 0.9ml (1ml total volume), into PS via PRV-PSV line. Then fills the syringe with air from the PRV. Mixes the lysate.
3. Move the diluted lysate into mixing coil.
4. Rinses the PSV-PRV-AIV/CBV line, bypasses mixloop (created samplePassF--based on samplePassCa)
5. Moves diluted lysate held in mixing coil to the CBV-AIV line. Primed to PRV.valve
6. Mixes diluted lysate with speDil (same protocol/ratio in loadMFBslug)
7. Positions slug in AIV for MFB (completes loadMFBslug)
Possible Valve Configuration Change for PRV::
GuSCN has to be primed to valve
Assumes that we don't change current DA reagent positions.
A. With DA reagents
1 AIV/CBV
2 ab1
3 speDil
4 guscn
5 Sipper // diluent?
6 waste2
7 ab2
8 air clean
If BAC arrays are performed on samples also for qPCR, then the sipper has to be converted into diluent.
B. w/o DA reagents (Nearly same as current PRV valve with MFB attached)
5/15/11 GuSCN is on PRV.3 Spare1: The valve configuration has not yet been altered.
1 AIV/CBV
2 diluent
3 guscn #was spare1
4 spare2
5 Sipper
6 waste2
7 spedil
8 air clean
def diluteMFBlysate #apart of PRVprocess.rb
# uses loadMFBslug above, but performs a dilution of the lysate for qPCR
# CAN PARAMETERIZE THE DILUTION???---Comes much later. Ask Brent how...
#Put in rinse of mixloop here.
loadMFBslug do
#Mixes lysate and GuSCN into syringe
Log.record "diluting Lysate in Syringe"
PSV.to :PTV
PTV.to :PRV
CBV.to :PRV
PRV.to :air
delay 2
PRV.to CBV
delay 2
PS.pull 0.1, PSslow
delay 5
PRV.to :guscn
PS.pull 0.9
delay 2
PRV.air
PS.fill
delay 2
#Moves diluted lysate to PRV mixing coil
Log.record "Parking diluted lysate in Mixing Coil"
PSV.to :mixloop
PTV.to :PRVmixing
PRV.to :CBV
PS.push MixLoopPull #need to determine volume start with MixLoopPull
delay 2
PSV.to waste1
PS.empty
PSV.to PTV
PTV.to PRV
#Rinses PRV-AIV-CBV line, bypasses mixloop
samplePassF
#Primes diluted lysate to PRV (CBV-AIV-line)
Log.record "moving diluted lysate into CBV-AIV line"
PSV.to :air
PS.fill
PSV.to :mixloop
PTV.to PRVmixing
PRV.to CBV
PS.push 0.9 PSslow
delay 2
PRV.waste2
PS.empty
#Mixes diluted lysate with speDil
Log.record "mixing lysate with speDil"
PSV.to :PML
CBV.to :PRV
PRV.to :air
delay 2
PRV.to :speDil
delay 2
PS.pull 0.225, PSslow
delay 5
PRV.to CBV
delay 2
PS.pull .250, PSslow
delay 5
end
end
def samplePassF cleanCS = nil #In samplePass.rb
#rinses PRV-AIV-CBV line, bypasses mixloop
#run option (flush) block afterwords
#also allows cleanCS protocol
Log.record "Beginning samplePassF"
CC.loadFlushPuck do
MFB.bypass if defined? MFB
#put 1.5ml of flush into PS
PSV.to PSR, :via=> :waste1
PSR.to :flush
PS.to 1.5
delay 5
PSV.to :waste1
PS.to 0
PSV.to PSR
PSR.to :air
PS.fill
#Backflush PRV - CBV line to CTV spit; bypasses mix loop
PRV.connect CBV
PSV.to :PTV, :via=> :air #Check this direction
CTV.to :air, :avoiding=>:intake
PTV.to :PRV
PS.to 0
#clear the line of fluid
PSV.to :air
PS.fill
PSV.to :PTV
PS.to 0
PSV.to :air
PS.fill
PSV.to :PTV
PS.to 0
rinseCS if cleanCS
yield if block_given?
end
end #samplePassF
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