User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/11

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Double digestion pUA66 XhoI / BamHI

Two new double digestion was set up to run overnight.

For a final volume of 50µl

  1. 30µl pUA66 plasmid
  2. 5µl 10xBSA
  3. 5µl 10xNEB#3
  4. 1µl XhoI
  5. 1µl BamHI

Tube 1 was placed in the PCR machine at 37ºC.

Tube 2 was placed in the incubator at 37ºC.

Gel Purification of digests from 09 of August 2010

This purification follows the digestion performed User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/09.

All 6 digestions from the pUA66 where purified by performing a cutting from the gel and then purifying the gel band.

  1. pUA66#1 = 300mg
  2. pUA66#2 = 400mg
  3. pUA66#3 = 290mg
  4. pUA66#4 = 330mg
  5. pUA66#5 = 340mg
  6. pUA66#6 = 240mg

The procedure was carried out in ration relations to the band weight. e.g. 300mg => 300µl in 1:1 and 300mg => 900µl in 1:3

  1. Add 1:3 ratio of QG buffer
  2. Dissolve band in 47ºC for 10 minutes by inverting every 2 minutes till all gel is liquified
  3. Add 1:1 ratio of isopropanol and mix well
  4. Apply solution to a QIAgen quick column
  5. Centrifuge for 1 minute at 13,000 rpm and discard flow-through
  6. Add 500µl of QG buffer
  7. Centrifuge for 1 minute and discard flow-through
  8. Add 750µl of PE wash with Ethanol
  9. Centrifuge for 1 minute and discard flow-through
  10. Centrifuge for an additional minute and discard flow-through
  11. Place the QIAgen column in an eppendorf tube
  12. Apply 30µl of water to the center of the column and wait for 1 minute
  13. Centrifuge for 1 minute to elute the purified digested plasmid

Note: If the DNA will be used for salt-sensitive applications, such as blunt-end ligation and direct sequencing, let the column stand 2–5 min after addition of Buffer PE, before centrifuging.

Gel verification

In order to check that the purified vectors retain the band and strength a gel was set up as follows

  1. Lane 1: 10µl 1kb ladder
  2. Lane 2: 2µl pUA66#1 BamHI/XhoI purified
  3. Lane 3: 2µl pUA66#2 BamHI/XhoI purified
  4. Lane 4: 2µl pUA66#3 BamHI/XhoI purified
  5. Lane 5: 2µl pUA66#4 BamHI/XhoI purified
  6. Lane 6: 2µl pUA66#5 BamHI/XhoI purified
  7. Lane 7: 2µl pUA66#6 BamHI/XhoI purified
  8. Lane 8: 2µl pUA66 - undigested

Gel Result: 12082010-digestion2xpurified-and-undigest-compare.jpg Note: If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.