Preparing Sanger Sequencing (Eurofins): Difference between revisions
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PreDec2022>EricMiller (Created page with "__FORCETOC__ ==Only PCR product or Plasmid== Used if Eurofins will be added a primer to the Sanger sequencing tube. *Add 10ul of DNA to a 1.5ml microcentrifuge tube. The concentration should be: PCR Products, Purified 0.100–0.300kb 10–20 ng/ul PCR Products, Purified 0.301–1.000kb 20–40 ng/ul PCR Products, Purified >1.000kb 40–60 ng/ul Plasmid, Prepared < 1 .000kb 30-60 ng/ul Plasmid, Prepared 1.0 << 6.0kb 60-150 ng/ul Plasmid, Prepared 6.0 <...") |
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However, don't sweat it if you have less than this concentration. Please dilute back if your concentration is larger than the high end of these values. | However, don't sweat it if you have less than this concentration. Please dilute back if your concentration is larger than the high end of these values. | ||
*Place a sequencing barcode on | *Place a separate sequencing barcode on each tube, and send Eric the barcode letters/numbers, as well as the contents of the sample or samples. Each sample gets a different barcode. | ||
*Put a thin bit of parafilm around to the tube, to prevent it from opening. | *Put a thin bit of parafilm around to the tube, to prevent it from opening. | ||
| Line 28: | Line 28: | ||
*Have Eric print the order slip (which goes into the envelope) as well as the shipping label. The shipping label gets cut out and taped on all 4 sides to the UPS envelope. | *Have Eric print the order slip (which goes into the envelope) as well as the shipping label. The shipping label gets cut out and taped on all 4 sides to the UPS envelope. | ||
*Once it is ready, put in the shipping mailbox behind the dining center, by the blue bus stop. It gets collected by ~5:30pm-6:00pm each day; keep samples in the lab refrigerator or -20 freezer if they are ready after this time. | *Once it is ready, put in the shipping mailbox behind the dining center, by the blue bus stop. It gets collected by ~5:30pm-6:00pm each day; keep samples in the lab refrigerator or -20 freezer if they are ready after this time. | ||
==Plasmid + primer, or PCR + primer== | ==Plasmid + primer, or PCR + primer== | ||
Same procedure, but use 5ul DNA. Add 5ul of a single primer at the same concentration as PCR -- a 1:10 dilution of the blue-capped, stock solution. | Same procedure, but use 5ul DNA. Add 5ul of a single primer at the same concentration as PCR -- a 1:10 dilution of the blue-capped, stock solution. | ||
==Key Primers== | |||
* If sequencing a P-80 derived CRISPR plasmid, use primer O-263 | |||
* If sequencing a P-52, pET28a(+) derived expression plasmid, use the T7 forward primer, provided by Eurofins. | |||
Latest revision as of 12:57, 16 December 2022
Only PCR product or Plasmid
Used if Eurofins will be added a primer to the Sanger sequencing tube.
- Add 10ul of DNA to a 1.5ml microcentrifuge tube. The concentration should be:
PCR Products, Purified 0.100–0.300kb 10–20 ng/ul
PCR Products, Purified 0.301–1.000kb 20–40 ng/ul
PCR Products, Purified >1.000kb 40–60 ng/ul
Plasmid, Prepared < 1 .000kb 30-60 ng/ul
Plasmid, Prepared 1.0 << 6.0kb 60-150 ng/ul
Plasmid, Prepared 6.0 << 20.0kb 150-250 ng/ul
However, don't sweat it if you have less than this concentration. Please dilute back if your concentration is larger than the high end of these values.
- Place a separate sequencing barcode on each tube, and send Eric the barcode letters/numbers, as well as the contents of the sample or samples. Each sample gets a different barcode.
- Put a thin bit of parafilm around to the tube, to prevent it from opening.
- Place all tubes in a small bag (see the top drawer, with the autoclave tape) and place in UPS shipping envelope (found in the bookcase in the write-up area).
- Have Eric print the order slip (which goes into the envelope) as well as the shipping label. The shipping label gets cut out and taped on all 4 sides to the UPS envelope.
- Once it is ready, put in the shipping mailbox behind the dining center, by the blue bus stop. It gets collected by ~5:30pm-6:00pm each day; keep samples in the lab refrigerator or -20 freezer if they are ready after this time.
Plasmid + primer, or PCR + primer
Same procedure, but use 5ul DNA. Add 5ul of a single primer at the same concentration as PCR -- a 1:10 dilution of the blue-capped, stock solution.
Key Primers
- If sequencing a P-80 derived CRISPR plasmid, use primer O-263
- If sequencing a P-52, pET28a(+) derived expression plasmid, use the T7 forward primer, provided by Eurofins.