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--- bdev:minithermocycler [2020/10/22 18:27]
richard
+++ bdev:minithermocycler [2021/08/16 22:22] (current)
@@ Line 1: / Line 1: @@
+Example data demonstrating the PCR works
+{{ :bdev:timestamp_95_8_60_15.png?400 |}}
+{{ :bdev:timestamp_95_15_60_30.png?600 |}}
+{{ :bdev:thermocycling_comparison.png?600 |}}
+Green channel was plotted to compare.
+Heat conditions:
+  * 95C 15 seconds
+  * 60C 30 seconds
+  * 40 cycles
+Samples
+  - beta-actin - PCR with taqman probe
+  - blank - water control with master mix
+Note: after 30 cycles signal was visable
+{{ :bdev:pcr_bactin_example.png?400 |}}
 Steps for building a mini thermocycler
@@ Line 7: / Line 41: @@
-Custom Parts:
+Custom Parts:{{ :bdev:thermoblock_holder_1inch_half.stl |}}
 |1. Milling thermoblock. It costs $100 to make this.  {{ :bdev:thermoblock_holder_1inch_half.stl |}}|{{ :bdev:image_52_.png?200 |}}|
 |2. Lid with 2 3mm set screws {{ :bdev:drillblock_1inch_lid_half.stl |}}| {{ :bdev:image_55_.png?200 |}}|
-|3. Block holder {{ :bdev:thermoblock_holder_1inch_half_1_.stl |}}|{{ :bdev:image_56_.png?200 |}}|
+|3. Block holder {{ :bdev:hermoblock_holder_1inch_half.stl |}}|{{ :bdev:image_56_.png?200 |}}|
 |4. Extrusion connects {{ :bdev:tslot_hinge_thermalblock.stl |}} | {{ :bdev:image_57_.png?200 |}}|
 |5. Brd base {{ :bdev:thermocycle_brd.stl |}}| {{ :bdev:image_58_.png?200 |}}|
 |6. PCB board {{ :bdev:pcbs_thermominiblock.zip |}} Parts: {{ :bdev:gerberfiles_parts.zip |}}| {{ :bdev:above_view.png?200 |}} {{ :bdev:bottom_view.png?200 |}}|
+|7. Single tip imager {{ :bdev:singletip_imager.stl |}}|{{ :bdev:singletip_imager.png?200 |}}|
-Off the shelf ($43) + assembled brd is around $20 a unit:
+Off the shelf ($43) + assembled brd is around $20 a unit + pipetters $20:
 |1. Power supply| {{ :bdev:powersupply.png?200 |}}|
 |2. Peltier cooling system | {{ :bdev:pelier_cooling_system.png?200 |}}|
@@ Line 24: / Line 59: @@
 |5. Heater/Thermistors| {{ :bdev:heater_thermistors.png?200 |}}|
 |6. Arduino Nano | {{ :bdev:arduino_nano.png?200 |}}|
+<code>
+import time,datetime,os
+from datetime import datetime
+import json,re
+import serial
+import subprocess
+def timestamp(gg):
+ now = datetime.now() # current date and time
+ date_time = now.strftime("%m/%d/%Y, %H:%M:%S")
+ print("date and time:",date_time)
+ gg.write("date and time: " + date_time)
+def openport(prt):
+  try:
+   ser = serial.Serial(prt, 115200, timeout=0.2)
+   time.sleep(2)
+  except:
+   print("its not connecting")
+  return ser
+def whatstheports():
+ try:
+  output = subprocess.check_output("python3 -m serial.tools.list_ports -v", shell=True).decode()
+  b = re.split("\n", output)
+  if len(b)>1:
+      for i in b:
+          if re.match('^ ', i):
+              pass
+          else:
+              try:
+               dada = (re.sub(' .*', '',i))
+               if re.match('^.*AMA0.*$', dada):
+                pass
+               else:
+                #ser = serial.Serial('/dev/ttyUSB0', 115200, timeout=0.2)
+                ser = serial.Serial(dada, 115200, timeout=0.2)
+                time.sleep(2)
+                ser.write(b'info\n')
+                time.sleep(0.2)
+                resp = (ser.readlines()[0].decode())
+                ser.close()
+                if re.match('^tempcontrol.*', resp):
+                    return dada
+              except:
+               pass
+  else:
+     print("You need to plug in the device")
+ except:
+  print("please plug the device in")
+def read():
+  port = whatstheports()
+  ser = openport(port)
+  ser.write(b'readlevel\n')
+  b = (ser.readlines())
+  temp = float(b[0].decode().rstrip())
+  print(temp)
+def incubate(stemp):
+  print("Beginning to incubate at this temp "+stemp)
+  print("To stop, ^c to kill the program")
+  port = whatstheports()
+  ser = openport(port)
+  ser.write(b'settemp '+stemp.encode()+b'\n')
+  ser.write(b'readlevel\n')
+  time.sleep(0.2)
+  b = (ser.readlines())
+  temp = float(b[0].decode().rstrip())
+  a = 1
+  while a >0:
+   ser.write(b'readlevel\n')
+   time.sleep(0.2)
+   b = (ser.readlines())
+   temp = float(b[0].decode().rstrip())
+   print(temp)
+def coolon():
+  print("cooling ... ")
+  port = whatstheports()
+  ser = openport(port)
+  ser.write(b'fanon\n')
+  time.sleep(0.5)
+  ser.write(b'coolon\n')
+  time.sleep(0.5)
+  ser.write(b'settemp 0\n')
+  a = 1
+  while a > 0:
+   ser.write(b'readlevel\n')
+   time.sleep(0.2)
+   b = (ser.readlines())
+   temp = float(b[0].decode().rstrip())
+   print(temp)
+def flashon():
+  port = whatstheports()
+  ser = openport(port)
+  ser.write(b'strobmic\n')
+  return ser
+def flashoff(ser):
+  ser.write(b'off\n')
+  ser.close()
+def runcycle(dtemp,dtemptm,etemp,etemptm,cy):
+  print(int(cy))
+  ccy = int(cy)
+  gg = open("log.txt", "w")
+  timestamp(gg)
+  gg.write("Thermcycler denature "+dtemp+"C time "+dtemptm+" extend "+etemp+" time "+etemptm+" cycle "+cy+"\n")
+  print("Thermcycler denature "+dtemp+"C time "+dtemptm+" extend "+etemp+" time "+etemptm+" cycle "+cy)
+  print("To stop, ^c to kill the program\n")
+  port = whatstheports()
+  ser = openport(port)
+  time.sleep(1)
+  #ser.write(b'setp 300\n')
+  for i in range(0,ccy):
+   print("cycle "+str(i))
+   gg.write("cycle "+str(i)+"\n")
+   cycle(dtemp,dtemptm,etemp,etemptm,cy,ser,i,gg)
+  print("finished thermocycling")
+  timestamp(gg)
+  gg.write("finished thermocycling\n")
+  gg.close()
+def cycle(dtemp,dtemptm,etemp,etemptm,cy,ser,cycle,gg):
+  print("Cycle "+str(cycle))
+  print("Setting denaturing temperature to "+str(dtemp)+"C")
+  gg.write("Cycle "+str(cycle)+"\n")
+  gg.write("Setting denaturing temperature to "+str(dtemp)+"C\n")
+  ser.write(b'settemp '+dtemp.encode()+b'\n')
+  ser.write(b'readlevel\n')
+  time.sleep(0.2)
+  b = (ser.readlines())
+  temp = float(b[0].decode().rstrip())
+  while temp < (int(dtemp)+1):
+   ser.write(b'readlevel\n')
+   time.sleep(0.2)
+   b = (ser.readlines())
+   temp = float(b[0].decode().rstrip())
+   print(temp)
+   gg.write(str(temp)+"\n")
+   if temp == (int(dtemp)):
+       break
+  time.sleep(int(dtemptm))
+  ser.write(b'settemp 0\n')
+  print("Setting extend temperature to "+str(etemp)+"C")
+  print("Cooling ... ")
+  gg.write("Setting extend temperature to "+str(etemp)+"C\n")
+  gg.write("Cooling ... \n")
+  ser.write(b'fanon\n')
+  time.sleep(0.3)
+  ser.write(b'coolon\n')
+  time.sleep(0.3)
+  ser.write(b'readlevel\n')
+  time.sleep(0.2)
+  b = (ser.readlines())
+  temp = float(b[0].decode().rstrip())
+  while temp > (int(etemp)+1):
+   ser.write(b'readlevel\n')
+   time.sleep(0.2)
+   b = (ser.readlines())
+   temp = float(b[0].decode().rstrip())
+   print(str(cycle) + ' ' + str(temp))
+   gg.write(str(cycle) + ' ' + str(temp)+'\n')
+   if temp == (int(etemp)):
+       break
+  ser.write(b'fanoff\n')
+  time.sleep(0.3)
+  ser.write(b'cooloff\n')
+  time.sleep(0.3)
+  print("extend temperature begins")
+  gg.write("extending \n")
+  time.sleep(0.3)
+  ser.write(b'settemp '+etemp.encode()+b'\n')
+  while temp > (int(etemp)+1):
+   ser.write(b'readlevel\n')
+   time.sleep(0.2)
+   b = (ser.readlines())
+   temp = float(b[0].decode().rstrip())
+   print(temp)
+   gg.write(str(temp)+"\n")
+  gg.write("incubating at extend temperaure\n")
+  print("incubating at extend temperaure\n")
+  time.sleep(int(etemptm))
+input_var = input("Thermobock controller [help|h, about|a, incubate|i, cycle|c], read|r, cool, flashon: ")
+if re.match("help|h", input_var):
+    print("This controller the thermocycler. Here are the available commands")
+    print("\thelp[h] - Provides the options")
+    print("\tabout[a] - About the program, the product its used for and the developer")
+    print("\tincubate[i] - Incubate at specific temperate. The command goes like this -incubate[i]_[your temperature]. So something like this 'i_55' or 'incubate_55' for incubating at 55C")
+    print("\tread[r] - Read temperature sensor, it reports in Celcius")
+    print("\tcycle - Cycling conditions. Argument goes like this -cycle[c] extent_time_denature_time_cycles. Like cycle_94_30_55_180_40 for 55C for 30 seconds then 55 for 180 seconds")
+    print("\tflashon - Turns on LED")
+elif re.match("port", input_var):
+    whatstheports()
+elif re.match("read|r", input_var):
+    read()
+elif re.match("cool", input_var):
+    coolon()
+elif re.match("incubate_.*|i_.*", input_var):
+    [arg, temp] = re.split('_', input_var)
+    incubate(temp)
+elif re.match("cycle_.*|c_.*", input_var):
+    [arg, dtemp,dtemptm,etemp,etemptm,cy] = re.split('_', input_var)
+    runcycle(dtemp,dtemptm,etemp,etemptm,cy)
+elif re.match("flashon", input_var):
+    ser = flashon()
+    floff = input("Press [enter] to turn off")
+    flashoff(ser)
+else:
+    print("command not recognized")
+</code>
+----
+<code>
+#include <Servo.h>
+#include <PID_v1.h>
+//#include <FastPID.h>
+Servo myservo;  // create servo object to control a servo
+#include <Wire.h>
+#include <SoftwareSerial.h>
+#define THERMISTOR_PIN A7
+#define TEMPCONTROL 3
+#define FANPIN 5
+#define THERMOELECTRIC_COOL 6
+#define LED 9
+//Define the aggressive and conservative Tuning Parameters
+double aggKp=150, aggKi=0.5, aggKd=0;
+int thermoelectricval = 255;
+int ledval = 255;
+int tempcontrol = 3;
+String command;
+float currpos;
+int celsius;
+int levelstreamon;
+double settemp = 0;
+double Setpoint, Input, Output;
+int tme = 250;
+//Specify the links and initial tuning parameters
+//PID myPID(&Input, &Output, &Setpoint, consKp, consKi, consKd, DIRECT);
+PID myPID(&Input, &Output, &Setpoint, aggKp, aggKi, aggKd, DIRECT);
+#define NUMTEMPS 20
+short temptable[NUMTEMPS][2] = {
+   {1, 841},
+   {54, 255},
+   {107, 209},
+   {160, 184},
+   {213, 166},
+   {266, 153},
+   {319, 142},
+   {372, 132},
+   {425, 124},
+   {478, 116},
+   {531, 108},
+   {584, 101},
+   {637, 93},
+   {690, 86},
+   {743, 78},
+   {796, 70},
+   {849, 61},
+   {902, 50},
+   {955, 34},
+   {1008, 3}
+};
+void setup() {
+  // put your setup code here, to run once:
+  Serial.begin(115200);
+  analogWrite(TEMPCONTROL, 0);
+  analogWrite(FANPIN, 0);
+  Setpoint = settemp;
+  //turn the PID on
+  myPID.SetMode(AUTOMATIC);
+  analogWrite(THERMOELECTRIC_COOL,0);
+  analogWrite(LED,0);
+}
+void loop() {
+   Input = read_temp();
+   double gap = abs(Setpoint-Input); //distance away from setpoint
+   myPID.SetTunings(aggKp, aggKi, aggKd);
+   myPID.Compute();
+   analogWrite(TEMPCONTROL,Output);
+   int rawvalue = analogRead(THERMISTOR_PIN);
+   float celsius = read_temp();
+   int fahrenheit = (((celsius * 9) / 5) + 32);
+  if (levelstreamon == 1) {
+   Serial.println(celsius);
+   delay(500);
+  }
+ if(Serial.available())
+ {
+    char c = Serial.read();
+    if (c== '\n')
+    {
+      parseCommand(command);
+      command = "";
+    }
+    else
+    {
+      command +=c;
+    }
+ }
+ delay(1);
+}
+float parseCommand(String com)
+{
+  if(com.equalsIgnoreCase("info")){
+    Serial.println("tempcontrol");
+  }
+  else if(com.equalsIgnoreCase("levelstreamon")){
+   levelstreamon = 1;
+  }
+  else if(com.equalsIgnoreCase("defaultpid")){
+    double aggKp=19.56, aggKi=0.71, aggKd=134.26;
+  }
+  else if (com.substring(0,4) == "setp") {
+    aggKp = com.substring(5).toDouble();
+  }
+  else if (com.substring(0,4) == "seti") {
+    aggKi = com.substring(5).toDouble();
+  }
+  else if (com.substring(0,4) == "setd") {
+    aggKd = com.substring(5).toDouble();
+  }
+  else if(com.equalsIgnoreCase("coolon")){
+   analogWrite(THERMOELECTRIC_COOL,thermoelectricval);
+  }
+  else if(com.equalsIgnoreCase("cooloff")){
+   analogWrite(THERMOELECTRIC_COOL,0);
+  }
+   else if (com.substring(0,10) == "setledval") {
+    ledval = com.substring(11).toInt();
+  }
+  else if(com.equalsIgnoreCase("levelstreamoff")){
+   levelstreamon = 0;
+  }
+  else if(com.equalsIgnoreCase("fanon")){
+   analogWrite(FANPIN, 255);
+  }
+  else if(com.equalsIgnoreCase("fanoff")){
+   analogWrite(FANPIN, 0);
+  }
+  else if(com.equalsIgnoreCase("readlevel")){
+     int rawvalue = analogRead(THERMISTOR_PIN);
+     float celsius = read_temp();
+     Serial.println(celsius);
+  }
+  else if (com.substring(0,12) == "setthermoval") {
+    //Serial.println("setting temperature");
+    thermoelectricval = com.substring(13).toInt();
+    //Serial.println(settemp);
+  }
+  else if (com.substring(0,7) == "settemp") {
+    //Serial.println("setting temperature");
+    settemp = com.substring(8).toInt();
+    //Serial.println(settemp);
+    Setpoint = settemp;
+  }
+  else if(com.equalsIgnoreCase("strobmic")){
+    strobingmic(ledval, tme);
+    delay(10);
+  }
+}
+float read_temp()
+{
+   int rawtemp = analogRead(THERMISTOR_PIN);
+   float current_celsius = 0;
+   byte i;
+   for (i=1; i<NUMTEMPS; i++)
+   {
+      if (temptable[i][0] > rawtemp)
+      {
+         float realtemp  = temptable[i-1][1] + (rawtemp - temptable[i-1][0]) * (temptable[i][1] - temptable[i-1][1]) / (temptable[i][0] - temptable[i-1][0]);
+         if (realtemp > 255)
+            realtemp = 255;
+         current_celsius = realtemp;
+         break;
+      }
+   }
+   // Overflow: We just clamp to 0 degrees celsius
+   if (i == NUMTEMPS)
+   current_celsius = 0;
+   return current_celsius;
+}
+void strobingmic(int ledval, int tme){
+ int strobtimer = 1;
+ while (strobtimer > 0 && Serial.available() == 0){
+  analogWrite(LED, ledval);
+  delayMicroseconds(tme);
+  analogWrite(LED, 0);
+  delayMicroseconds(tme);
+  strobtimer++;
+ }
+ analogWrite(LED, 0);
+}
+</code>

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