User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/19

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Checked plates The plates I grew from yesterday User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18#Plasmid Transformation (pUA66katE#A1#3 and pUA66katE#A1#4) and User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18#Grow pUA66katE#A1 both grew. The colonies on plate pUA66katE#A1#3 looked similar to the original plate pUA66katE#A1 and indicator that this sample contains the insert. I had the digestions already running. Gel 2%, insert screening To screen the remaining colonies from pUA66katE#A1 ran overnight digestions on a 2% gel, User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18#Overnight Digestion (pUA66katE#A1#3 and pUA66katE#A1#4) pUA66katE#A1#2 Serie digested ( First XhoI for 7 hour and then BamHI for 12 hours) pUA66katE#A1#3 Double digest, XhoI/BamHI, 2 hours pUA66katE#A1#4 Double digest, XhoI/BamHI, 2 hours Gel 2% Add 1.2g Agarose powder Add 60ml 1XTAE Heat in microwave until no specs and cool Add 2µl Ethidium Bromide Pour in prepared tray Loading: Lane 1: 10µl, 100bp NEB Ladder Lane 2: 60µl, pUA66katE#A1#2, XhoI/BamHI Lane 3: 50µl, pUA66katE#A1#3, XhoI/BamHI Lane 4: 50µl, pUA66katE#A1#4, XhoI/BamHI Lane 5-8: Blank Gel Picture: Analysis: None of the colonies screened shows the insert. At this point I have screened all four colonies. I have detected one potential candidate but I did not take a sample and will need to redo a screening for all colonies to verify. Prepared ladder 100bp I prepared a new 100bp NEB ladder by adding ( where did I put this protocol?) 20µl of Miniprep - overnight growth (67 | pUA66katE#5) I used 2 bottles out of a total of 6 for miniprep, based on the overnight growth User:Howard Boland/Notebook/Art from Synthetic Biology/2010/08/18#Overnight Growth (pUA66katE and plasmid 67). Spin all bottles for 10 minutes in centrifuge Discard supernatant Store 4 of the bottles at -20ºC (Two of the pUA66katE and one pBestLuc) Place the remaining 2 bottles in an icebucket for miniprep For each of the 2 (pUA66katE#5 and p67) remaining bottles: Resuspend in 500µl cold P1 buffer Distribute 250µl in two eppendorf tubes For each tube add 250µl P2 Lysis buffer and wait 4 minutes Add 350µl N3 neutralisation buffer Centrifuge at 13,000 rpm for 10 minutes Apply the first tube to a QIAgen Quick column Centrifuge for 1 minute at 13,000 rpm and discard flow-through Apply the second tube to the column Centrifuge for 1 minute at 13,000 rpm and discard flow-through, all the plasmids is now bound to the column For each column add 500µl PB wash buffer Centrifuge for 1 minute at 13,000 rpm and discard flow-through For each column add 750µl PE with Ethanol final wash buffer Centrifuge for 1 minute at 13,000 rpm and discard flow-through Centrifuge for an additional minute at 13,000 rpm and discard flow-through Place columns in each labelled Eppendorf tubes Add 30µl of water to the centre of the column and wait for 1 minutes Centrifuge for 1 minute at 13,000 rpm to elute plasmid Store plasmids in an icebucket (or in the freezer at-20ºC) Digestion - (67 | pUA66katE#5) I prepared a master mix to digest each of the 30µl plasmid to a final volume of 50µl Master mix (total volume 40µl) 1µl 100xBSA Buffer 10µl 10xNEB#3 Buffer 2µl XhoI 2µl BamHI 25µl Water Reaction Aliquote 20µl Master mix to each of the tubes Place in incubator at 37ºC for 2 hour Gels 1% and 2%, vector extraction and insert screening Prepare a 2% gel to screen for insert. Prepare a 1% gel to cut out p67 2% Gel Add 1.2g Agarose Powder Add 60ml of 1xTAE Buffer Heat using a microwave until all specks have cleared Pour in tray 1% Gel Add 0.6g Agarose Powder Add 60ml of 1xTAE Buffer Heat using a microwave until all specks have cleared Pour in tray Loading 2% Lane 1: 10µl of 100b NEB Ladder Lane 2: 50µl of pUA66katE#5, XhoI/BamHI Lane 3-8: BLANK Loading 1% Lane 1: 10µl of 100b NEB Ladder Lane 2: 50µl of p67, XhoI/BamHI, cut Lane 3-8: BLANK Gel Picture 1% (extract 67) Gel Picture 2% (pUA66katE#5, new ligation plate) Overnight Growth I prepared 4 bottles of 10ml LB Broth and add 10µl of 50mg/µl Kanamycin stock. A proper screening of the pUA66katE#A1 plate was performed by labelling each colony. As these colonies had been picked before by swabbing and now regrown I check that none of the smears overlapped. 4 Bottles were setup and I picked 4 separate and labelled both plate and tube The bottles were placed in a shaker at 37ºC overnight SAP and Ligation A key problem with my vector is that there seems to be non-specific binding resulting in self-ligation. This prompted me to return to the use of SAP (Shrimp Alkaline Phosphate) disrupt the binding sites and prevent self-ligation. Some mistakes were made during this process. My vector has a concentration of around 15ng/µl, thus in 30µl I have a total of 15ng/µl*30µl = 450ng ~ 0.5µg of DNA. The SAP is used in 1unit/µg of DNA. So I will be using 0.5µl of SAP. SAP Reaction, final volume will be 50µl. 0.5µl SAP 30µl DNA (pUA66 XhoI/BamHI) 5µl 10xSAP Buffer 14.5µl Water Place in incubator for 15minutes at 37ºC Place in a 65ºC water bath for 15 minutes to terminate reaction I did the same reaction for a total of 4 samples from my box. Ligations Four ligations were then setup using all SAP 1µl Ligation Buffer 1µl T4 50µl SAP reaction 7µl katE XhoI/BamHI I setup two reactions in a standard ligation 1:7 1µl Ligation Buffer 1µl T4 1µl pUA66 XhoI/BamHI 7µl katE XhoI/BamHI All ligations were incubated overnight at 37ºC