====== PCR ======

===== State of the art PCR  and electrophoresis =====

Since 2025, we have restarted our legendary efforts to have reproducible PCR results. Running workflow includes flowbox, sterile equipment, new centrifuges and thermocyclers... and of course university-grade knowledge.

==== Bio-Rad iCycler image conventer ====

The iCycler software is storing images of 96-wells fluorescence activities in their own binary blob .ISI without the possibility to export to any standard format like TIFF or DICOM. So one have to hexdump and write their own conventer...

<code c isi2tiff.c>
// Convert BIO-RAD ISI to TIFF
// 16bit grayscale little-endian
// CC-BY-NC sachy@brmlab

#include <stdio.h>
#include <stdlib.h>
#include <string.h>
#define ISIWIDTH 342
#define TIFFWIDTH 310
#define TIFFHEIGHT 240
#define TIFFHEAD {0x49,0x49,0x2a,0x00,0x88,0x8a,0x04,0x00}

unsigned char tt[]={0x11,0x00,0x00,0x01,0x03,0x00,0x01,0x00,
0x00,0x00,0x36,0x01,0x00,0x00,0x01,0x01,0x03,0x00,0x01,0x00,0x00,0x00,0xf0,0x00,
0x00,0x00,0x02,0x01,0x03,0x00,0x02,0x00,0x00,0x00,0x10,0x00,0x10,0x00,0x03,0x01,
0x03,0x00,0x01,0x00,0x00,0x00,0x01,0x00,0x00,0x00,0x06,0x01,0x03,0x00,0x01,0x00,
0x00,0x00,0x01,0x00,0x00,0x00,0x11,0x01,0x04,0x00,0x02,0x00,0x00,0x00,0x72,0x8b,
0x04,0x00,0x12,0x01,0x03,0x00,0x01,0x00,0x00,0x00,0x01,0x00,0x00,0x00,0x15,0x01,
0x03,0x00,0x01,0x00,0x00,0x00,0x02,0x00,0x00,0x00,0x16,0x01,0x03,0x00,0x01,0x00,
0x00,0x00,0x80,0x00,0x00,0x00,0x17,0x01,0x04,0x00,0x02,0x00,0x00,0x00,0x6a,0x8b,
0x04,0x00,0x1a,0x01,0x05,0x00,0x01,0x00,0x00,0x00,0x5a,0x8b,0x04,0x00,0x1b,0x01,
0x05,0x00,0x01,0x00,0x00,0x00,0x62,0x8b,0x04,0x00,0x1c,0x01,0x03,0x00,0x01,0x00,
0x00,0x00,0x01,0x00,0x00,0x00,0x1d,0x01,0x02,0x00,0x02,0x00,0x00,0x00,0x2d,0x00,
0x00,0x00,0x28,0x01,0x03,0x00,0x01,0x00,0x00,0x00,0x02,0x00,0x00,0x00,0x52,0x01,
0x03,0x00,0x01,0x00,0x00,0x00,0x01,0x00,0x00,0x00,0x53,0x01,0x03,0x00,0x02,0x00,
0x00,0x00,0x01,0x00,0x01,0x00,0x00,0x00,0x00,0x00,0x48,0x00,0x00,0x00,0x01,0x00,
0x00,0x00,0x48,0x00,0x00,0x00,0x01,0x00,0x00,0x00,0x00,0x6c,0x02,0x00,0x80,0x1e,
0x02,0x00,0x08,0x00,0x00,0x00,0x08,0x6c,0x02,0x00};

int main(int argc,char **argv)
{
	FILE *in=NULL;
	FILE *out=NULL;
	switch(argc)
	{
		case 2:
		{
			in=fopen(argv[1],"r");
			out=stdout;
			break;
		}
		case 3:
		{
			in=fopen(argv[1],"r");
			out=fopen(argv[2],"w");
			break;
		}
		default:
		case 0:
		case 1:
		{
			printf("isi2tif infile [outfile]");
			return -1;
			break;
		}
	}

	unsigned char buff[ISIWIDTH*2];
	unsigned char buff2[ISIWIDTH*2*2];
	unsigned char th[]=TIFFHEAD;
	unsigned short h=0;
	memset(buff,0x00,ISIWIDTH*2);
	fread(buff,1,38*2,in);

	fwrite(th,1,sizeof(th),out);
	while(fread(buff,2,ISIWIDTH,in)==ISIWIDTH && h++<TIFFHEIGHT)
	{
		memset(buff2,0xFF,sizeof(buff2));
		for(int i=0;i<sizeof(buff)/2;i++)
			memcpy(buff2+i*4,buff+i*2,2);

		fwrite(buff2,2*2,TIFFWIDTH,out);
	}
	fwrite(tt,1,sizeof(tt),out);

	fclose(in);
	fclose(out);
	return 0;
}
</code>


FIXME TODO Vic textu


===== Ye olde PCR and electrophoresis =====

DNA electrophoresis using the OpenPCR [[http://openpcr.org/]]

=== Status ===
  * openPCR working, dry run successful (SW: [[https://github.com/cathalgarvey/OpenPyCR]], running.)
  * Taq/dNTP PCR mix obtained from [[http://www.openbiotech.com]]
  * electrophoresis working, tested at hackathon
  * got chemicals:
    * TAE buffer [[http://letters.cathalgarvey.me/cargo-cults-and-electrophoresis/]])
    * ddH2O
    * PCR grade agarose (quite little, expensive)
    * EtBr
    * SYBR Gold (thanks [[user:Mrkva]] and [[user:barney]]!)
    * some DNA fragments for experimentation (pTracer plasmids)
  * freezer (-20°C) for DNA/enzyme storage is ready
  * we have a wonderful power supply (up to 300V) thanks to Tomsuch
  * transilluminator built from blue LEDs 

the first test run with crude agar, self made TAE, DIY loading dye (cresol red+sucrose), 60V (with the power source from hwlab), 1.5h:

{{:project:biolab:img_6511.jpg?200|}}


{{:project:biolab:img_6512.jpg?200|}}


second test run with agarose and several restriction digest samples using a strong laser and SYBR gold for visualization - blue light filter from infra-soldering station:

{{:project:2802_e4dd_960.jpeg?nolink&300 |}}


some notes for using SYBR gold: 
  * DO NOT STORE IN GLASS CONTAINER
  * protocol: [[https://tools.lifetechnologies.com/content/sfs/manuals/mp11494.pdf]] 

**first test run of the openPCR**  

Template: pTracer plasmid

Primers: inf pTracer F + R (10uM) designed by [[user:chido]] to amplify the GFP/Bsd gene 

PCR mix:
  dH2O 33ul
  mastermix Openbiotech 5x 10 ul
  primer F 2ul
  primer R 2ul
  template 1ng/ul 3ul
 total volume 50 ul

Thermocycler settings:
  95 °C 60 sec
    95 °C 30 sec
    60 °C 30 sec
    72 °C 60 sec
  x30
  4 °C 20 sec 

SYBR gold gel stain:
  30 ml TAE, 0.3g agarose gel
  stained in 10ul SYBR gold diluted in 100ml TAE, 40 min 

Results: 

We obtained the desired product using Openbiotech master mix that has been stored in -20 °C, but there was no product at all when using a mastermix that has been stored in the fridge. We aliquoted both SYBR gold gel stain and the PCR mastermix so thawing the stock solution is not necessary. 
OpenPCR works fine! Pictures and analyzed gel coming soon. Next step: devise a protocol for DNA isolation that can be used as a PCR template.

Useful links:
  * primer design guide: [[http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html]]
  * paper on horses/rats/cockroaches detection in food via PCR [[http://journals.tubitak.gov.tr/veterinary/issues/vet-07-31-3/vet-31-3-3-0601-30.pdf]]   
  * there are online companies that will synthetize custom primers for a fee [[http://www.operon.com]][[http://www.biogen.cz]]