Team:Michigan/Notebook
Notebook
February
-Recruited new members to MSBT
-Began project design
March
-Finalized project design
-Competed in OptiMize Social Innovation Challenge, won $11,000 grant to fund summer research
April
-Began OptiMize summer fellowship, got entrepreneurial training and learned about bench to bedside process. See https://www.optimizemi.org for more details.
May
-Hunted for and found lab space for the summer, and ordered supplies
-Trained new MSBT members in the lab
June
-Hosted WISE GISE camp, taught middle school girls about synthetic biology. See (LINK TO THAT PART OF HUMAN PRACTICE) for more details.
- 6/14/16: Made LB/cam plates, Made LB/cam broth, transformed pSB1C3BBa_K564012 from the 2014 kit into DH5a, and plated 50uL and 500uL of diluted transformed cells
- 6/15/16: little growth, started overnight culture from the one colony that grew, did another transformation of pSB1C3BBa_K564012 from the 2016 kit into DH5a, plated 50uL and 250uL of undiluted transformed cells
- 6/16/16: minipreped pSB1C3BBa_K564012 from overnight culture, one colony grew on plate from yesterday’s transformation, another colony grew on the plate from 6-14, started overnight cultures of each
- 6/17/16: Transformed pSB1C3 (high copy number plasmid) from 2016 kit into DH5a, used all 10uL and plated 50uL and 200uL undiluted, minipreped pSB1C3BBa_K564012 from yesterday’s overnight cultures
- 6/20/16: Nothing grew all weekend from pSB1C3 transformation from 6-17
- 6/27/16: Digested 10uL of pSB1C3BBa_K564012 from each of the minipreps from 6-17 with XbaI and PstI using NEB double digest calculator to develop protocol. (10uL DNA, 5uL 10X NEB buffer 3.1, 1uL XbaI, 1uL PstI, nuclease free water to 50uL total-> 37C for 15mins, 80C for 20mins, 4C infinite hold)
- 6/28/16: Made 50X TAE buffer, Made 10mg/mL ethidium bromide, Ran gel with restriction digest products from 6-27 (Ladder/Green/Red/Purple/Yellow), Imaged gel and posted to slack. Uploaded file to google docs in lab notebook folder
- 6/29/16: Resuspended primers to 40uM concentration (its the lowest concentration that could fit in the vial), Ran PCR of Red miniprep from yesterday’s restriction digest using primers designed to add EcoRI and HndIII sites before and after lacZ while isolating lacZ from the rest of the plasmid. Ran Blank (B) and two samples (1 and 2).
- 6/30/16: Ran 10uL of PCR products on 1.5% gel,ul, Cut out band with product and did gel extraction using NEB kit (20uL elution volume), put it in the freezer
July
No lab access, focussed on modeling and human practice
August
- 8/20/16: Putting lacZ into pet28a(+)… PCR of the lacZ plasmid using “EcoRI and XbaI lacZ F” and “HindIII PstI lacZ R”... Cut vector pet28a(+) with EcoRI and HindIII... Run PCR product on a gel with cut vector. Cleanup pcr product and cut vector. Cut PCR product with EcoRI and HindIII. Heat inactivate at 95C for 10 min. Cleanup cut PCR product... Ligate vector and insert and transform...Pick colonies and mini prep. Pet28a+ with only LacZ omega fragment: Amplify pet28a+ plasmids with LacZ with primers that will delete the 11-41 residues (“LacZ deletion R” and “LacZ deletion F”)...Ligate (KLD reaction)...Transform into highly competent cells Putting lacZ omega frag into submission vector: Order reverse primer which is complementary to C terminus of lacZ and has SpeI and PstI... Amplify lacZ omega frag using new primer containing SpeI and PstI and also “EcoRI and XbaI lacZ F”... Cut vector RFC10 with XbaI and SpeI... Run PCR product on a gel with cut vector. Cleanup pcr product and cut vector. Cut PCR product with XbaI and SpeI. Heat inactivate at 95C for 10 min. Cleanup cut PCR product... Ligate vector and insert and transform... Pick colonies and miniprep.
- 8/25/16: PCR purified the LacZ PCR(Ecori/HindiIII)
- 8/26/16: Digest of PCR product with EcorI and HindiIII, Run products on a 1% agarose gel, did not see any bands, Run pet28a and LacZ PCR product with EcoRI/HindiIII overnight. Set up two reactions for each plasmid
September
9/4/16 and 9/5/16
Miniprep of plasmids (30 min) and then digest 300 ng with XbaI and NotI (1.5 hr) then run a gel to see which colonies worked (45 min). All colonies appear to be empty vector :/ Digesting the cut vector even more by adding 25 ul cut vector (out of the 50 ul elution volume after gel extraction) + 3.5 ul hindIII + 3.5 ul EcoRI + 3.5 ul NEB Buff 2.1 Also digested the lacZ PCR product (redoing) by adding 25 ul PCR product (out of the 50 ul elution volume after gel extraction) + 3.5 ul hindIII + 3.5 ul EcoRI + 3.5 ul NEB Buff 2.1
9/7/16
Digested LacZ pcr product, pet 28 and pet28 digest (Ecori/HindiIII) with EcorI and HindiIII.
- 3.5uls buffer
- 3.5uls Ecori-HF
- 3.5uls HindiIII
- 25uls DNA
Run digest at 37C for one hour and 45 min, heat in
Loaded 42uls of the pet28 digest products, only digest of digest products showed up.Gel extracted pet28 vector 5kb~ and cleaned up with NEB gel purification kit.
- Phosphotase the two pet28 gel extractions
- 5uls antartic phosphotase
- 2uls Phosphotase buffer
- 5uls DNA
- 8uls nuclease free water
- Ligated vector and insert
9/27/16
Cutting plasmids with Not1 should yield a 3 kb fragment and a 5.3 kb fragment. If it only yields a 5 kb fragment then the insertion was not successful. Using two different tubes of NotI at the same time in case one is bad.
Protocol:
For each tube of DNA:
-1 uL NotI HF tube 1
-1 uL NotI HF tube 2
-4 uL DNA
-1 uL cutsmart buffer
-3 uL H2O
Incubate at 37C for 1 hour (started 10:12 pm, ended at ~11:30) then run on 1% agarose gel.
*The 1:9 digests actually just got 2uL of NotI from tube 1 because tube 2 ran out.
9/29/2016
Did PCR of lacZ plasmid and ran it on a gel (analytical gel not preparative) and uploaded image to drive. Still need to do a PCR cleanup of the PCR product. Pet28a plasmid is in Liu lab. Need to bring some of it to the USB. Took ligation product from 9/7 and digested with SalI then transformed. This gave 2 colonies which were cultured and minipreped. Some of these plasmids was then digested with NotI (which should yield a 5.3 kb frag and 3 kb frag), but this gave only one band which was ~7-10 kb. This might mean that the ligation worked but the NotI digest didn’t? We should digest these with HindIII and EcoRI (which should yield a 3 kb fragment and a 5.3 kb fragment if ligation worked).
October
-Cannot find lacZ deletion r or lacZ deletion f in the USB or Liu lab after looking extensively. Reordering but need to come up with an alternative method of showing that part of our project works.
-Ideas: Do PLA and NASBA then digest with DNase I and run agarose gel with SYBR gold. Protein gel to detect alpha fragment. Could be improved if we had dna coding for alpha frag. Possibly even just detecting PLA directly on a gel if it is sensitive enough? It looks like as little as 16.7 nM of probe 1 linked to probe 2 (short terminator) would be detectable on an agarose gel with sybr gold (40 ng in a 20 ul reaction). The M.W. of probe 1 linked to probe 2 (short terminator) is 95652 g/mol, M.W. of probe 2 (short terminator) is 45123, M.W. of probe 1 is 50467, M.W. of 3C connector is 5742, M.W. of 2C connector is 5453, M.W. of 1C connector is 5163... For testing PLA using agarose gel and sybr gold only… We want to shoot for ~200 ng of probe 1 linked to probe 2 (short terminator) product for maximum visibility on the gel. We want an equimolar amount of thrombin, probe 1, probe 2, and the connector. They should all be 0.1 uM final conc. We want 74.03 ng of human alpha thrombin because M.W. of thrombin is 37000 daltons. Our thrombin stock is 7.9 mg/ml so we need to dilute a small part of this stock (diluted in 1X T4 DNA ligase buffer) by a factor of 100 so that we can use 0.937 uL of that diluted stock in a 20 ul reaction. In this reaction, we will also need 100.95 ng of probe 1 and 90.24 ng of probe 2 and 11.48 ng 3C connector or 10.91 ng of 2C connector or 10.33 ng of 1C connector. We will also want 2 ul of T4 DNA ligase and 2 ul of T4 DNA ligase buffer per rxn. Incubate at 37C for ? minutes. Heat samples to 70C for 10 min to heat inactivate ligase and melt connector-probe interactions, then immediately load onto 1.75% agarose gel. Run a ladder and also run 100.95 ng probe 1 alone.
- 6/14/16: Diluting all oligos to 100 uM stocks except for connectors which are 200 uM... PLA probe 1: 40 uls H2O = 100uM, PLA probe 2: 40uls H2O = 100uM, PLA linker 1C:829uls H2O = 200uM, PLA linker 2C:680uls H2O = 200uM, PLA linker 3C:632uls H2O = 200uM, Dilutions of probes and linkers to use in the solutions… Dilute a portion of all stocks 100x
- 6/14/16: Ran ligation reaction first attempt. Did not see evidence of ligation.
- 6/14/16: 10/12/16: transformed pSB1C3 into dh5a
- 6/14/16: 10/15/16: picked colonies, started cultures. Got sequencing results for lacZ in pet28, confirmed that colonies 1, 3, 4, and 5 are good (not sure about others). Found LacZ deletion F and LacZ deletion R primers. Started PCR with these primers
- 6/14/16: 10/16/16: minipreped pSB1C3, digested with EcoRI and PstI, ran gel (1.5%). Attempted ligation reaction second time. Still no evidence of ligation.
- 6/14/16: 10/18/16: Attempted ligation reaction third time. Still no evidence of ligation. Cloning LacZ omega fragment into PSB1C3 submission vector: PCR of potential plasmids with the alpha fragment deletion using EcoRI and XbaI LacZ/SpeI LacZ R primers with annealing temperature at 72C using Phusion Digest.
