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#Overnight growth 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: Add 700µl of overnight growth culture broth to an Eppendorf tube 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 Resuspend each bottle in 500µl of P1 buffer Distribute each tube into 2 tubes each containing 250µl. For each tube add 250µl P2 buffer, wait 4 minutes For each tube add 350µl N3 buffer, invert 12 times Centrifuge all tube for 10 minutes at 13,000 rpm Apply first tubes of same batch (e.g. pUA66katE#A1-1 - 250µl) to a QIAgen quick column Centrifuge column for 1 minutes at 13,000 rpm and remove supernatant Apply second tubes of same batch (e.g. pUA66katE#A1-2 - 250µl) to the same QIAgen quick column Centrifuge column for 1 minutes at 13,000 rpm and remove supernatant, now the total 500µl is bound to each column For each column (4 in total) add 500µl PB Wash Centrifuge column for 1 minutes at 13,000 rpm and remove supernatant For each column (4 in total) add 750µl PE Wash with Ethanol Centrifuge column for 1 minutes at 13,000 rpm and remove supernatant Centrifuge column for an additional minutes at 13,000 rpm and remove supernatant Place column in a labelled eppendorf tube Apply 30µl of water to the centre of the tube and wait for 1 minute 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 Add 12µl 10xNEB#3 Add 1.2µl 100xBSA Add 4µl XhoI Add 4µl BamHI Add 38.8µl water Reaction To 15µl DNA add 15µl Master mix 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 1.2g Agarose powder 60ml 1xTAE Heat in microwave until no specs and cool Add 2µl Ethidium Bromide Pour in tray with comb Loading Lane 1: 10µl, 100bp NEB Ladder Lane 2: 50µl, kate1 XhoI/BamHI Lane 3: 50µl, kate2 XhoI/BamHI Lane 4: 50µl, kate3 XhoI/BamHI Lane 5: 50µl, kate4 XhoI/BamHI 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 katE2, 220mg katE3, 210mg katE4, 243mg Gel purification katE XhoI / BamHI Add 1:3 Volume QG Buffer katE2, 660µl katE3, 630µl katE4, 729µl Place in 47ºC water bath for 10 minute, mix ever 2 minute to dissolve gel Add 1:1 Volume Isopropanol katE2, 220µl katE3, 210µl katE4, 243µl Apply each tube to a separate column Centrifuge for 1 minute at 13,000 rpm, discard supernatant Apply remainder ( it was getting a bit full) for each tube to each related column Centrifuge for 1 minute at 13,000 rpm, discard supernatant Add 500µl QG Buffer Centrifuge for 1 minute at 13,000 rpm, discard supernatant Add 750µl PE Final wash with Ethanol Centrifuge for 1 minute at 13,000 rpm, discard supernatant Centrifuge for an additional minute at 13,000 rpm, discard supernatant Place columns in labelled eppendorf tubes Apply 30µl water to the center of each columns and wait for 1 minute 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: Lane 1: 10µl 1kb NEB Quick Ladder Lane 2: 50µl pUA66katE#A1, Digest XhoI/BamHI Lane 3: 50µl pUA66katE#A2, Digest XhoI/BamHI Lane 4: 50µl pUA66katE#B1, Digest XhoI/BamHI Lane 5: 50µl pUA66katE#B2, Digest XhoI/BamHI 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#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: Lane 1: 5µl 1kb NEB Quick Ladder Lane 2: 5µl katE#2, Digest XhoI/BamHI, Purified Lane 3: 5µl katE#3, Digest XhoI/BamHI, Purified Lane 4: 5µl katE#4, Digest XhoI/BamHI, Purified Lane 5: 5µl pUA66#1, Digest XhoI/BamHI Lane 6: 5µl pUA66#2, Digest XhoI/BamHI Lane 7-8: BLANK Gel Picture (taken 16 AUG 2010)