28S PCR #6 7/23/19
E53 E54 P13 P14 P15 P16 E46
Procedure
28S is also known as ITS2_Coral and is a ITS2 primer - Primer info
Samples E53 E54 P13 P14 P15 P16 E46 were used in this PCR (polimerase chain reaction) reaction
First A Master Mix of water, 2x mytaq mix, 5.8S-436 Primer (concentration: 10 micromolar), 28S-663 Primer (concentration: 10 micromolar)
| Component | Volume for 7 reactions (+5% error) |
|---|---|
| Water | 66.15 micoleters |
| 2x mytaq mix | 91.9 micoleters |
| 5.8S-436 Primer (concentration: 10 micromolar) | 7.35 micoleters |
| 28S-663 Primer (concentration: 10 micromolar) | 7.35 micoleters |
| BAS (Borine Serum Albumin) | .5 microliter |
24 micoleters of this master mix was added to 14 tubes For the 2 negative controls 12.5 micoleters water and 12.5 micoleters mytaq mix were added this was the first 5.8S.38 PCR so no positive control was available
1 microleter of the DNA samples was added to the coresponding tube
The samples were then placed in the thermocycler set to
| Step | Temperature | Duration | cycles |
|---|---|---|---|
| 1 | 92°C | 3:00 | 1 |
| 2 | 92°C | 0:30 | 25 |
| 3 | 56°C | 1:00 | 25 |
| 4 | 72°C | 0:15 | 25 |
| 5 | 72°C | 5:00 | 1 |
| 6 | 4°C | hold | - |
these amplified DNA samples were then tested by making a gel
to create the Gel the following steps were taken
1.5g agarose and 75ml 1X TAE buffer were mixed to make a 2% agarose 75ml gel mix the mix was microwaved and swirled until there were no flakes then 1 microliters Gelgreen was added
the mix was left to cool for 5 minutes then poured into the gel mold the gel was left to harden for 30 minutes
once hardened enough Used TAE buffer was poured into the gel box to cover the mold
5 microliter aliquots of DNA samples E53 E54 P13 P14 P15 P16 E46 and the two controls were made 1 microliter of 5x loading dye was added to each
First 3 microliters of the HyperLadder 75bp was added to the first well of the mold then the DNA samples were each added to the wells in the following order
** the second row on this Gel was used to retest some already run samlples to test if they work **
| Ladder | E53 | R54 | P13 | Negative Control | P14 | P15 | P16 | E46 | Positive Control (E5) |
| Ladder | E34 | R35 | Negative Control | E36 | E37 | Positive Control (E5) |
The voltage was set to 100V and run for 60 minutes
This is the resulting Gel
Notes
retrying E34-E37 did not work