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

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Overnight growth - successful
All bottles from the overnight growth User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13 worked great!

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

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

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)

Cut
 * 1) katE2, 220mg
 * 2) katE3, 210mg
 * 3) katE4, 243mg

Gel purification katE XhoI / BamHI

 * 1) Add 1:3 Volume QG Buffer
 * 2) katE2, 660µl
 * 3) katE3, 630µl
 * 4) katE4, 729µl
 * 5) Place in 47ºC water bath for 10 minute, mix ever 2 minute to dissolve gel
 * 6) Add 1:1 Volume Isopropanol
 * 7) katE2, 220µl
 * 8) katE3, 210µl
 * 9) katE4, 243µl
 * 10) Apply each tube to a separate column
 * 11) Centrifuge for 1 minute at 13,000 rpm, discard supernatant
 * 12) Apply remainder ( it was getting a bit full) for each tube to each related column
 * 13) Centrifuge for 1 minute at 13,000 rpm, discard supernatant
 * 14) Add 500µl QG Buffer
 * 15) Centrifuge for 1 minute at 13,000 rpm, discard supernatant
 * 16) Add 750µl PE Final wash with Ethanol
 * 17) Centrifuge for 1 minute at 13,000 rpm, discard supernatant
 * 18) Centrifuge for an additional minute at 13,000 rpm, discard supernatant
 * 19) Place columns in labelled eppendorf tubes
 * 20) Apply 30µl water to the center of each columns and wait for 1 minute
 * 21) 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



Gel Result 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)

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 and 2 pUA66-X-B from yesterday User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/13.

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)


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