User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/09

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Result Overnight Growth

Two bottles lost their caps and were discarded

IMG 0902.jpg


Overnight growth resulted in 8/10 10ml bottle with growth. 2 bottles had lost their caps during the shaking as were discarded. Out of the kept bottles: 6 bottles contained pUA66 2 bottles contained pUA66katE (Ligation test)

All bottles were spun down for 10 minutes with the supernatant discarded. For each bottle I:

  1. Added 500µl of P1 buffer and transferred 250µl to two eppendorf tubes
  2. For each eppendorf tube (8x2=16 in total) I added 250µl of Lysis buffer and waited 4 minutes
  3. For each eppendorf tube I then added 350µl of N3 buffer
  4. All tubes we spun at 13,000 rpm for 10 minutes
  5. I then took half the tubes belonging together and for each transferred into a QIAGEN quick column
  6. Centrifuge for 1 minute at 13,000 rpm and discard supernatant
  7. I repeated step 5. and 6. for the remaining tubes
  8. I then washed each column with 500µl of PE Wash and centrifuged for 1 minute
  9. For each column I added 750µl of PE Final Wash and centrifuged for 1 minute
  10. To remove ethanol residue I then centrifuge for an additional 2 minutes
  11. I placed each column in eppendorf tubes, added 50µl of water and waited for 1 minute
  12. I span the columns with the tube for 2 minutes to elute the plasmid DNA

The 10 tubes where each labelled => End of Miniprep


Using the samples from the miniprep I moved onto checking the concentration

  1. pUA66#1; 13.42 ng/µl; 2.06 260/280
  2. pUA66#2; 15.46 ng/µl; 1.94 260/280
  3. pUA66#3; 15.70 ng/µl; 2.16 260/280
  4. pUA66#4; 14.49 ng/µl; 2.13 260/280
  5. pUA66#5; 16.67 ng/µl; 2.05 260/280
  6. pUA66#6; 18.08 ng/µl; 1.97 260/280
  1. pUA66katE#1; 15.84 ng/µl; 1.99 260/280
  2. pUA66katE#2; 17.96 ng/µl; 2.07 260/280

Digestion with XhoI

For a final volume of 50µl

  1. 5µl 10xBSA
  2. 5µl 10xNEB#3
  3. 1µl XhoI
  4. 30µl DNA
  5. 9µl Water

For 8 reaction I made up a mastermix for the above using

  1. 40µl 10xBSA
  2. 40µl 10xNEB#3
  3. 8µl XhoI
  4. 36µl Water

Mixed and then aliquoted 20µl of this into of 8 reaction tubes containing 30µl DNA.

The reaction took place for 2 hrs @ 37ºC in an incubator

Digestion with BamHI

Following on from the XhoI digest I then made another mastermix using

  1. 40µl 10xBSA
  2. 40µl 10xNEB#3
  3. 8µl BamHI

Mixed and then aliquoted 11µl of this into of 8 reaction tubes from the previous digest.

The reaction took place for 2 hrs @ 37ºC in an incubator

Agarose Gel Extraction

Three gels were prepared using 0.6g of Agarose powder with 60ml of water, heated until no specks remained and then cooled. 2µl of Ethidium Bromide was added to the gel and 3 double lanes were made using autoclaved tape to connect the comb incisions, the gel was then loaded as follows:

Gel A: Checking cloning of katE into pUA66, a positive result would produce a ~400bp product

  1. Lane 1: 5µl 1kb Ladder
  2. Lane 2: 5µl Digest pUA66katE#1, XhoI/BamHI
  3. Lane 3: 5µl Digest pUA66katE#1, XhoI/BamHI
  4. Lane 4: Blank
  5. Lane 5: Blank
  6. Lane 6: 5µl Digest pUA66katE#2, XhoI/BamHI
  7. Lane 7: 5µl Digest pUA66katE#2, XhoI/BamHI
  8. Lane 8: Blank

Gel B:

  1. Lane 1: 10µl 1kb Ladder
  2. Lane 2-3: pUA66-1 XhoI/BamHI
  3. Lane 4-5: pUA66-2 XhoI/BamHI
  4. Lane 6-7: pUA66-3 XhoI/BamHI
  5. Lane 8: Blank

Gel C:

  1. Lane 1: 10µl 1kb Ladder
  2. Lane 2-3: pUA66-4 XhoI/BamHI
  3. Lane 4-5: pUA66-5 XhoI/BamHI
  4. Lane 6-7: pUA66-6 XhoI/BamHI
  5. Lane 8: Blank

Gel Documentation

Gel A: 09082010-pUA66katE-BamHI-XhoI3.jpg

Gel B: 09082010-pUA66-Gel1-Cut.jpg

Gel C: 09082010-pUA66-Gel2-Cut.jpg