User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/14

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Contents

Overnight growth - successful

All bottles from the overnight growth User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13#Overnight growth worked great! Image:IMG_4212.jpg

Glycerol Stock

As these cultures may contain the insert I decided to take out glycerol stock of two of the tubes, pUA66katE#A1 and pUA66katE#B2, for each I did the following:

  1. Add 700µl of overnight growth culture broth to an Eppendorf tube
  2. Add 300µl of 50% Glycerol

At the moment I only had access to a -20ºC freezer - I will move it into -80ºC at first opportunity.


Miniprep

All bottles were centrifuged, and supernatant removed. Of the total batch of 12 bottles - 3 from each plate - 4 tubes were selected for miniprep: pUA66katE#A1, pUA66katE#A2, pUA66katE#B1, pUA66katE#B3. The other bottles were placed in the freezer -20ºC.

The miniprep was setup as follows

  1. Resuspend each bottle in 500µl of P1 buffer
  2. Distribute each tube into 2 tubes each containing 250µl.
  3. For each tube add 250µl P2 buffer, wait 4 minutes
  4. For each tube add 350µl N3 buffer, invert 12 times
  5. Centrifuge all tube for 10 minutes at 13,000 rpm
  6. Apply first tubes of same batch (e.g. pUA66katE#A1-1 - 250µl) to a QIAgen quick column
  7. Centrifuge column for 1 minutes at 13,000 rpm and remove supernatant
  8. Apply second tubes of same batch (e.g. pUA66katE#A1-2 - 250µl) to the same QIAgen quick column
  9. Centrifuge column for 1 minutes at 13,000 rpm and remove supernatant, now the total 500µl is bound to each column
  10. For each column (4 in total) add 500µl PB Wash
  11. Centrifuge column for 1 minutes at 13,000 rpm and remove supernatant
  12. For each column (4 in total) add 750µl PE Wash with Ethanol
  13. Centrifuge column for 1 minutes at 13,000 rpm and remove supernatant
  14. Centrifuge column for an additional minutes at 13,000 rpm and remove supernatant
  15. Place column in a labelled eppendorf tube
  16. Apply 30µl of water to the centre of the tube and wait for 1 minute
  17. To elute, centrifuge tube with column for 1 minutes at 13,000 rpm

2xDigestion on minipreps

In order to verify that the insert was present a double digestion with XhoI and BamI was needed. I decided to set up a 30µl digest for each of the minipreps, the cost of this is 15µl DNA leaving me with half.

Master mix (4x)

This master mix will have a total volume of 60µl which distributed into 4 bottle results in 15µl each

  1. Add 12µl 10xNEB#3
  2. Add 1.2µl 100xBSA
  3. Add 4µl XhoI
  4. Add 4µl BamHI
  5. Add 38.8µl water

Reaction

  1. To 15µl DNA add 15µl Master mix
  2. Place in incubator for 2 hours at 37ºC

Gel extraction of digested katE, PCR product

This gel extraction is based on the digest setup previous night Editing User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13#Digestion of Purified katE product

2% Gel

  1. 1.2g Agarose powder
  2. 60ml 1xTAE
  3. Heat in microwave until no specs and cool
  4. Add 2µl Ethidium Bromide
  5. Pour in tray with comb

Loading

  1. Lane 1: 10µl, 100bp NEB Ladder
  2. Lane 2: 50µl, kate1 XhoI/BamHI
  3. Lane 3: 50µl, kate2 XhoI/BamHI
  4. Lane 4: 50µl, kate3 XhoI/BamHI
  5. Lane 5: 50µl, kate4 XhoI/BamHI
  6. Lane 6-8: Blank

Gel Result Image:IMG_4238.jpg

Error: I seem to have lost katE1 for some reason not sure why...proceeding with the other ones - I am a bit suspicious about Lane 4, katE3, as it is slightly higher - digestion problem?

Gel PICTURE (TAKEN 16 AUG 2010) Image:14082010-purification-kate.jpg


Cut

  1. katE2, 220mg
  2. katE3, 210mg
  3. katE4, 243mg

Gel purification katE XhoI / BamHI

  1. Add 1:3 Volume QG Buffer
    1. katE2, 660µl
    2. katE3, 630µl
    3. katE4, 729µl
  2. Place in 47ºC water bath for 10 minute, mix ever 2 minute to dissolve gel
  3. Add 1:1 Volume Isopropanol
    1. katE2, 220µl
    2. katE3, 210µl
    3. katE4, 243µl
  4. Apply each tube to a separate column
  5. Centrifuge for 1 minute at 13,000 rpm, discard supernatant
  6. Apply remainder ( it was getting a bit full) for each tube to each related column
  7. Centrifuge for 1 minute at 13,000 rpm, discard supernatant
  8. Add 500µl QG Buffer
  9. Centrifuge for 1 minute at 13,000 rpm, discard supernatant
  10. Add 750µl PE Final wash with Ethanol
  11. Centrifuge for 1 minute at 13,000 rpm, discard supernatant
  12. Centrifuge for an additional minute at 13,000 rpm, discard supernatant
  13. Place columns in labelled eppendorf tubes
  14. Apply 30µl water to the center of each columns and wait for 1 minute
  15. To elute, centrifuge for 1 minute at 13,000 rpm and keep tube

Running Gel of 2xDigested ligased minipreps

Final step to check if the plasmid contains the insert.

The gel was loaded as follows:

  1. Lane 1: 10µl 1kb NEB Quick Ladder
  2. Lane 2: 50µl pUA66katE#A1, Digest XhoI/BamHI
  3. Lane 3: 50µl pUA66katE#A2, Digest XhoI/BamHI
  4. Lane 4: 50µl pUA66katE#B1, Digest XhoI/BamHI
  5. Lane 5: 50µl pUA66katE#B2, Digest XhoI/BamHI
  6. Lane 6-8: BLANK

Running at 100V, 400mA, 60 minutes

Image:IMG_4241.jpg

Gel Result Image:IMG_4248.jpg Nah! Selfligation - and we are back to square one :( Well not entirely - at least I will check if I can use the new insert and vector - which have hopefully been digested and purified well.

Gel Picture (taken 16 AUG 2010) Image:14082010-1prcent-pUA66kat-2xdigest.jpg

Running Gel of purified products

This gel contains 4 purified katE-X-B from today User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/14#Gel purification katE XhoI / BamHI and 2 pUA66-X-B from yesterday User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13#Digestion of Miniprep.

The gel was loaded as follows:

  1. Lane 1: 5µl 1kb NEB Quick Ladder
  2. Lane 2: 5µl katE#2, Digest XhoI/BamHI, Purified
  3. Lane 3: 5µl katE#3, Digest XhoI/BamHI, Purified
  4. Lane 4: 5µl katE#4, Digest XhoI/BamHI, Purified
  5. Lane 5: 5µl pUA66#1, Digest XhoI/BamHI
  6. Lane 6: 5µl pUA66#2, Digest XhoI/BamHI
  7. Lane 7-8: BLANK

Gel Picture (taken 16 AUG 2010) Image:14082010-1percent-pUA66-2xdigest-katE2x-digest.jpg


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