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.

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…

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;
}

TODO Vic textu

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

• 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 Mrkva and 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:

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:

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 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.comhttp://www.biogen.cz