Technical information
Emma Jones; Michelle Durrant; and Sarah Noble-Longster
Prep notes
Day 2:
| ITEM | AMOUNT | NOTES |
| PER PAIR | ||
| Digsol | 300 μl
(need 250 μl) |
At room temp |
| Proteinase K | 25 μl
(need 20 μl) |
On ice
(but do not freeze at any stage) Bought as a suspension from Qiagen (19131 or 19133); concentration not stated |
| 1M DTT | 10 μl
(need 5 μl) |
On ice |
| 4M ammonium acetate | 400 μl
(need 300 μl) |
At room temp |
| 100% ethanol | 1 x glass universal
(need 2 ml) |
At room temp |
| Liquid waste beaker | 1 | |
| 10 mM Tris pH 8 | 30 μl
(need 25 μl) |
Kit elution buffer (e.g. NE, EB) spiked with 0.2 ng/μl pUC18-mtcat
On ice |
| Primer: JHmtF3 (100 μM ) | 3 μl
(need 1 μl) |
On ice |
| Primer: JHmtR3 (100 μM ) | 3 μl
(need 1 μl) |
On ice |
| Q5 Hot-Start polymerase master mix | 15 μl
(need 10 μl) |
On ice |
| PER BAY | ||
| 0.5 ml eppendorfs | 1 box per bay
(need 1 tube per pair) |
|
| Cat hair in a universal | 1 per bay
(need 3 strands per pair) |
|
| Tweezers (next to cat hair) | 1 per bay | |
| EQUIPMENT | ||
| Hotblocks at 55 oC | ||
| OTHER | ||
| Sponges on ice per bay by the sinks, to collect their PCR reactions (1 tube per pair) | Run their samples in the PCR machine after each session
(use programme ‘L2 Genetics: cat hair’ – see below for details). Freeze when complete |
Digsol
Per 100 ml: 4 ml 0.5 M EDTA pH 8 (–> 20 mM)
0.685 g NaCl (–> 120 mM )
5 ml 1 M Tris-HCl pH 8 (–> 50 mM)
81 ml dH2O
Autoclave
Add:
5 ml 20% SDS (–> 1%)
pH to 8 with NaOH
0.22 μm-filter into a sterile receptacle, then store at room temperature
Primers
JHmtF3: 5’-GATAGTGCTTAATCGTGC-3’
JHmtR3: 5’-GTCCTGTGGAACAATAGG-3’
PCR programme
1 x 94 oC / 5 min
30 x 94 oC / 30 s
54 oC / 30 s
72 oC / 40 s
1 x 72 oC / 10 min
Day 3:
| ITEM | AMOUNT | NOTES |
| PER PAIR | ||
| DraII
(NEB: R0503S – called EcoO109I but need labelling as DraII) Supply undiluted |
1 μl
(need 1 μl) |
On ice |
| BsaAI
(NEB: R0531S) Diluted 1 in 5 in Diluent C (B8003S) |
1 μl
(need 1 μl) |
On ice |
| BsmAI
(NEB: R0529S) Diluted 1 in 5 in Diluent B (B8002S) |
1 μl
(need 1 μl) |
On ice |
| NEB CutSmart buffer | 5 μl
(need 3 μl) |
On ice |
| See below for expected PCR/digest results | ||
| PER BAY | ||
| Gel equipment for 2 gels per bay | 2 x 250 ml conical flasks (with 25 ml flasks in the top)2 x 100 ml cylinder2 x gel trays2 x 20-well combs1 x 2 L beaker for collecting TAE |
Students get gel tanks and
lane sheets out themselves |
| Midori Green | Tube per bay | Put out with sign telling them not to throw away the tube |
| 100bp-Plus GeneRuler | Tube per bay | Put out with sign telling them not to throw away the tube |
| EQUIPMENT | ||
| Hotblocks at 37 oC | ||
| Waterbaths at 52 oC | ||
| OTHER | ||
| 1 x tank of 1X TAE available in lab | ||
| Return their PCR samples from the previous session | On ice | |
