Team:UIUC-Illinois/Notebook/LabNotebook

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Revision as of 02:31, 4 October 2012

Week 4 Notes

6/18/2012

- Made our own NEB Buffer 2
- RNA scaffold ready for synthesis
- PCR attempt 3: Using hotstart mix at 63 degrees, using protet as the template DNA and including a blank tube that contained no template DNA as a control. Gel reveals another failed PCR with all primer dimers.

6/19/2012

- Made digestions of Petrobrick and K12 cells using EcoRI as restriction enzyme; Ran a gel; brightest band was at 5 kb mark.
- Made more 1x TAE buffer
- Designed primers for BBa_K157006 restriction enzyme site (KpnI/XhoI) incorporation in order to put into pETDuet-1 along with BBa_K157005.
- Created a plan to subclone WT PUF into pBAD (inducible by IPTG) and eYFP into protet.
- Designed primers for the WT PUF with Angela, who submitted them for ordering.
- Inoculated E0030 from glycerol stock for the PCR tomorrow as well as MC4100.

6/20/2012

- Primers and RNA Scaffold are ordered.
- Will put in a pETDuet-1 subculture later today so miniprep of the plasmid and digestion/ligation can start tomorrow of BBa_K157005.
- IDT responded saying the RNA scaffold can't be synthesized through gBlock synthesis. They suggested synthesis through Ultramer Oligos.
- Miniprepped MC4100 and E0030 (stored both in the temp box of the -20).
- Performed PCR with E0030 (eYFP) as the template to make 2 tubes of control (no PUF binding site) and 2 tubes with the PUF binding site. Used the hotstart mix and diluted the primers from 30 mM to 10 mM. Gel bands of 700-800 base pairs confirmed a successful PCR. PCR cleanup was performed, and the resulting DNA put into the tem pbox of the -20.

6/21/2012

- Miniprepped the pETDuet-1.
- Digested both pETDuet-1 and BBa_K157005 with EcoRI and PstI in EcoRI buffer.
- Emailed Dr. Silver about how they synthesized their scaffold and will base decision upon her reply.
- Preliminary split-GFP/PUF tethering conceptualization done.
- Digested the 2 minipreps of control eYFP, the 2 minipreps of PUF eYFP, pBAD, and ptet.
- Ran digest and will view gel tomorrow.

6/22/2012

- The plan is to start growing cells in TB by Monday or Tuesday so that we can prepare M9 with Glucose. However, will need to test procedures with blank K12 and then with petrobrick-K12 so that we have a control.
- Double digested BBa_K157005 and pETDuet-1 with EcoRI and PstI in NEB EcoRI buffer. Ran two samples on a gel.
- Ran 2 gels of 1 kb ladder: pET duet and RID0015 and pBAD with ptet. The second gel was a 1 kb ladder with control eYFP 1, control eYFP 1, PUF eYFP 1, PUF eYFP 2.

Week 5 Notes

6/25/2012

- Started making TB media. Added tryptone, yeast, glycerol to make media, autoclaved. Need to add a 100 mL filter of .17 M KH2PO4 and .72 M K2HPO4. After that, need to grow 5 mL of the cells (both normal and petrobrick-transformed) in the TB along with 1:1000 ratio of antibiotic (CM) and grow it overnight.
- Extracted BBa_K157005 and pETDuet-1 from the gel. pETDuet-1 had a very faint band.
- The wrong ladder was used for BBa_K157005 so the band was not located.
- Digests were run again with NEB Buffer 3 and 2 uL of enzymes were used to cut the pETDuet-1 vector.
- Scaffold sent for synthesis through IDT's miniGene option.
- Ran gel to see if digests worked, all were successful, but not pET duet.
- Ran digests through the nanodrop and got low numbers for all.
- Went ahead and did ligation to make the following: pBAD+control eYFP, pBAD+PUF eYFP, ptet+control eYFP, ptet+PUF eYFP.
- Transformed the ligations into both DH5a and BL21.

6/26/2012

- Finished making the TB media.
- Prepared (2) 5 mL cultures of K12-Petrobrick in the TB.
- An additional two innoculations of normal K12 were prepared to grow overnight.
- Heard back from UW's team advisor from last year, Ingrid Swanson-Pultz, and she replied back with what strains of E. Coli they used. We will be fine using K12 cells because she said there wasn't much difference from the alkane production with the different cells. We will not need to order any more.
- Need to order C15 Alkanes to inject in one of the production cultures to ensure alkane production.
- Digestion of BBa_K157005 failed again with no presence of bands.
- Digestion of pETDuet-1 worked so the BBa_K157005 plasmid was run on a gel to confirm viability. The plasmid was still viable so double digestion was done again with EcoRI-HF/PstI-HF in NEB Buffer 4 with BSA and Alkaline Phosphatase.
- Transformations of DH5a and BL21 resulted in 3 lawns and no growth. A replate was necessary, and they were streaked out.
- Performed additional transformation of all the following into DH5a: pBAD+control eYFP, pBAD+PUF eYFP, ptet+control eYFP, ptet+PUF eYFP.

6/27/2012

- Both the TB cultures were successful. However, one of the normal K12 innoculations did not grow.
- Therefore, made two more TB cultures using just the K12 innoculation which worked.
- In addition, prepared CM and Amp plates for future use.
- Replates of pBAD with control/PUF eYFP in both DH5a and BL21 all had growth. However, BL21 cells looked translucent and flat. Inoculated all of the plates. No growth on transformations of the DH5a pBAD/ptet YFP constructs, so replated all of them. Checked resistance of ptet, and it's Cm, not Amp.
- Started biobricking PUF WT.
- Ran two PCR reactions with hotstart mix at 61 and 63 degrees C.
- Ran gel of PUF PCR samples done in May, bands looked to be the correct size. Gel extracted all five samples.
- Inoculated PSB1C3 overnight culture.

6/28/2012

- Made the M9 Media.
- The TB cultures from the K12 innoculations were successful.
- Currently, all cultures that we plan to use are in the 4 degree room. We will not take further action on the production protocol until we have received the C15 alkanes and Dr. Jin is back.
- We have all the materials to start the production media culture, but we will need Dr. Jin's guidance on the extraction method as well as GCSM analysis as the last steps of the protocol happen rather immediately. We will wait until next week to finish it up.
- Digestions of BBa_K157005 and pETDuet-1 seemed to have worked. However, the gel showed the split-gfp band at 300bp even though it was expected to be ~250bp.
- Overnight inoculations: the DH5a inoculations of pBAD with control and PUF eYFP both grew. However, both of the BL21 inoculations didn't grow at all.
- The replates of the YFP constructs didn't grow at all. So retransformed BL21 with WT PUF + control eYFP and WT PUF + PUF eYFP.
- Also retransformed DH5a with pBAD that had both control and PUF eYFP as well as the same in ptet.
- Digested the PSB1C3 culture made 6/27/12.
- Digested the PSB1C3, then ran a gel of the 3 total PUF PCR samples and 2 PSB1C3 miniprepped sample. Both PSB1C3 showed two bands, which was not expected (one at about 2kb and one a 1kb).
- Will have to run a control uncut PBSB1C3 with digested PSB1C3 to check what the problem is, could be that the strain used for inoculation had an insert in it.
- The PUF PCR digests looked about right.
- Gel extracted all 3 PUF PCR samples and the one band from the PSB1C3 that was at the right size.
- Started an overnight culture of PSB1C3.

6/29/2012

- Today more M9 media was made as we were unaware that it is not suppose to be autoclaved.
- Did a miniprep of pSB1C3
- None of the eYFP constructs in DH5a grew.
- Conclude that neither the control eYFP of the PUF eYFP constructs were properly ligated within either pBAD or ptet.
- While the retransformations of BL21 grew some odd looking colonies, it is almost certain that they only contain the WT PUF plasmid and not a correct eYFP plasmid.
- Attempt a second ligation, used same old digest (because the bands on that were correct).
- Ran the ligation overnight.
- Inoculated the successful but weird looking and dubious BL21 plates.

Week 6 Notes

7/2/2012

- Over the weekend: checked inoculations and had no growth on the control eYFP, so reinoculated it. It proceded to fail again.
- Ran a 24 hour ligation of pBAD/ptet YFP constructs. Plated them, they failed again.
- Checked transformations of DH5a with ptet+control/PUF eYFP. Had a few small colonies! so inoculated.
- Did another ligation using Angela's new procedure.
- Also did inoculation of 10 tubes of DH5a for making competent cells.

7/3/2012

- Ran a gel for PCR samples (control and PUF/YFP)
- Inoculations of YFP in protet grew, so made glycerol stock, miniprepped, and stored in the temp box in the -20. Also nandropped them.
- Did a colony PCR of the colonies with protet and control YFP or protet and PUF binding site YFP. Diluted the primers for this from 30 mM to 10 mM.
- Colony PCR fails.
- Digested PSB1C3 with EcoRI and PstI in buffer 2. Digested for 2 hours.

7/5/2012

- Made 10 tubes of DH5a competent cells.
- Streaked out Control YFP and PUF binding site YFP from glycerol stock.
- Looked at the gel from the 7/2/12 digestions of PSB1C3. There were two bands again. Both PSB1C3 glycerol stocks have the same problem. The stocks could have been incorrectly prepared. Perhaps there was an insert in the stocks.
- Will likely just use linearized PSB1C3 from biobrick kit for ligation.
- Made 1L LB and autoclaved some more 1.5mL microfuge tubes.

7/6/2012

- Double digestion of BBa_K157005/pETDuet-1 was ran again with EcoRI-HF and PstI-HF in NEB Buffer 4 with BSA for 4 hours. Digestion failed as neither the pETDuet-1 was present, nor was there presence of the split-CFP band.
- Digestion was ran again with creation of a supermix in order to assure correct pipetting under the same conditions except for 3 hours. Digestion failed once more, no presence of pETDuet-1 and extremely faint split-CFP band.
- pETDuet-1 and BBa_K157005 were both inoculated from glycerol stocks in order to miniprep more plasmid for further digestion.
- Digestion will be tried once more with vector concentrations of 500ng and 1000ng along with addition of Alkaline Phosphatase to see if there is any increase in yield.
- BBa_K157007 (split-cYFP) was transformed as well in order to be used if digestion of the BBa_K157005 part doesn't succeed.
- Streaked out plates all grew successfully. Performed colony PCR on them, but had an annealing time of 30 sec instead of 1 minute.
- Got primers for WT PUF in pBAD, diluted to 2.5*10^-5.
- Did PCR of the WT PUF for insertion into pBAD. Used hotstart mix, DMSO, and 2 minute annealing step.
- Ran colony PCR and WT PUF PCR on the same gel, got all primer dimers in every lane. Need to redo both.
- PCR'ed two more WT PUF samples.
- Started the following overnight cultures: 2 of the ISB 179 PSB1C3, 2 of the 6/4/12 prep PSB1C3, 1 of the Protet, 1 of the E0030 (YFP), two WT PUF.
- Transformed PSB1C3 from the biobrick kit 1 into DH5a, plated it as well.

7/7/2012

- Inoculated BBa_K157005 and pETDuet-1 to make new volume stocks of plasmids.
- Concurrently plated BBa_K157005 and pETDuet-1 in case the inoculations from glycerol stocks don't work and so I can inoculate from colonies if needed.
- Plated BBa_K157007 (the split-cYFP part) in case it works better than the BBa_K157005 split-cCFP part.
- However, colonies did not grow even after plated two plates as a backup.
- Miniprepped BBa_K157005 and pETDuet-1 inoculations put in last night and got very low, unusable concentrations (perhaps due to inadequate incubation time).
- Miniprepped pETDuet-1 and BBa_K157005 from this morning and got higher concentrations.
- Double digested BBa_K157005/pETDuet-1 with EcoRI-HF/PstI-HF with BSA and Alkaline Phosphatase for 3 hours (BBa_K157005 was digested both at 500ng and 1000ng vector quantity).
- Gel purified both bands which seemed to appear better than previous attempts. Concentrations after purification were still fairly low so chances of successful ligation might be compromised.
- Redid PCR of WT PUF with a temp gradient of 59-63-67-71. Used the supermix.
- Redid colony PCR with a temp gradient of 59-63-67-71 and the colony PCR supermix.
- Inoculated BBa_157005.
- Ran a gel of the WT PUF for pBAD. Only the 2 lanes showed any bands at all - the ladder and the 59 degree lane (had the right band!)
- Redid another gel with more dye, but still saw nothing else.
- Ran a gel of the colony PCR and got all primer dimers.
- PCR cleanup on the successful 59 degree lane for WT PUF, yielded a nanodrop of 63.7 ng/uL.
- Digested the pBAD miniprep and the WT PUF from the PCR.
- Ran a gel of the digestion with high melt gel. Got bands of the right length, but WT PUF was very faint.
- Used procedure for gel extraction, yielded very low nanodrop! Did ligation anyways
- PCR'ed four samples of WT PUF for subcloning into pBAD.
- Ran a gel of the PCR products. Only the 59 and 71 degrees samples worked (63 and 67 didn't).
- PCR cleanup of the two samples that worked, and digested with HindIII and EcorI.
- Gel extracted the two bands, then ligated over night.

Week 7 Notes

7/8/2012

- Inoculated BBa_K157005 and pETDuet-1 from bacterial cultures grown the day before.
- Ligated BBa_K157005 with the digested pETDuet-1 plasmid with a 10:1 ratio of insert to plasmid along with a no insert control.
- Miniprepped inoculations of BBa_K157005 and pETDuet-1 grown from bacterial cultures and received high concentrations (it seems inoculation from bacterial colonies is best).
- The ligations were then transformed into DH5a cells with 5uL of vector and the transformations were plated after 6.5 hours of incubation.
- Inoculations were made of BBa_K157005 and pETDuet-1 from bacterial cultures in order to prepare for more samples if needed.
- Work with the split-CFP construct will be suspended due to limitations of time.
- Made glycerol stock of pBAD 30 inoculations, stored in green box in neg 80.
- Miniprepped 2 tubes of pBAD and nanodropped.
- Stopped ligation after 8 hours and did 20 min inactivation at 80 degrees in the PCR machine.
- Transformed the ligations, so WT PUF in pBAD in DH5a on Amp plates.
- Inoculated pETDUET and K175005.

7/9/2012

- Started growing the K12-petrobrick cultures in the M9 media for Alkane production
- Decided to suspend work on the split-CFP integration into pETDuet-1 and focus on sending a PUF-CFP sequence out for synthesis.
- Tried to made plates (LB, Amp, Amp/Cm) but didn't have enough Agar in the mix, so they didn't solidify. Need a recipe other than that in notes.
- Starting from scratch for YFP by redoing the PCR. Used DMSO and supermix.. Failed, getting ghost bands that are very faint and at the incorrect length.
- PCR'ed three more samples of WT PUF for subcloning into pBAD

7/10/2012

- Incubation day; waiting on C15 alkanes to arrive.
- Made Amp, CM, and Amp-CM plates
- Transformed last ligation of PUF.
- Miniprepped inoculations of pBAD30.
- Yesterday the PCR of YFP failed, so redid with different temps on melting and annealing temps. used E0030 as new template, and no hotstart polymerase.
- Tried both the control site and PUF binding site at GC buffer and HF buffer.
- Ran a gel, all worked
- Performed PCR cleanup, got good nanodrop numbers.
- Digested all PCRs and protet with EcoRI and HindIII. Gel of digestion shows success.
- Performed gel purification, but got concentrations all under 6 ng/uL.
- Tried several ligations of control and PUf binding site YFP into protet anyways. Used 10:1 insert to vector ratios. Running ligations overnight.
- Did PCR cleanup on the three samples from yestereday, digested with EcorI and HindIII.
- Digested pBAD as well.
- Ran gel of the digests, only one of the PCR's worked, the digest of pBAD didn't work.
- Extracted the PCR sample digest that worked, and ligated it with a previously digested pBAD sample

7/11/2012

- Incubation for alkane production finished; took out the culture from the chromotography tubes; Not sure whether we extracted sample using Ethyl Acetate or not.
- Talked to Dr. Jin about GCMS; He directed us to Dr. Lee. He also mentioned that before GCMS, the C15 alkanes have to be injected in M9 media to test for a standard curve
- Transformed ligations, and plated on CM plates.
- Plated 4 pTET transformations, placed back in 37 degree room.
- Poured LB into 50mL conical tubes in sterile hood in MMG since somehow previous LB got contaminated.
- Transformed and plated the ligation reaction

7/12/2012

- Designing primers for cCFP amplification, PUF-CFP tethering of both N and C termini, and creating check primers for MSC1 and MSC2 of pETDuet-1 in order to sequence the constructs.
- Transformations from yesterday failed, so replated all of them.
- Started a second ligation from the digests, now using a 6:1 insert:vector ratio instead of a 10:1.
- There were no colonies, replated two more plates from the 7/10/12 digest.
- Also transformed and plated two plates using a ligation from 7/7/12

7/13/2012

- Found many colonies, however the number of colonies seems to be too many. Difficult to isolate individual colonies.

@@ Line 47: / Line 47: @@
                      <li><a name="week6puf" >Week 6</a></li>
                      <li><a name="week7puf" >Week 7</a></li>
-                     <li><a name="week8puf" >Further Documentation</a></li>
+                     <li><a name="week8puf" >Week 8</a></li>
+                    <li><a name="week9puf" >Further Documentation</a></li>
              </div>

5/29/2012	- Designed 9 primers for LacI. LacZ. and eYFP. Prepared LB. - Inoculation of B0014.J23100.wtPUF. B0030.Q04121 - Transformed the psB1C3 biobrick into DH5a cells
5/31/2012	- Transformed C0012, I732005, E0030, I732017. Also transformed PSB1A3, PSB1AK3, PSB1K3. - Inoculated LB media with a psB1C3 colony from the plate grown the day before. - Met with Prof. Bhalerao to talk about PUF application and project direction (RNA scaffolding).
6/1/2012	- Short-term goal changed: make one testing construct, [rbs-puf-eYFP] cloned into P.protet plasmid with a ptet promoter and terminator - Began inoculation of C0012, I732005, E0030, I732017, PSB1A3, PSB1AK3, PSB1K3. Was incubated for 9 hours, then put in 4 degree room - Inoculated again due to the round-bottom tube being misplaced. Mini-prepped the inoculation.
6/2/2012	- Cultured [PUF+PIN], E0030, and P. protet. designed check primers for [rbs-puf-eYFP]
6/3/2012	- Inoculated with a psB1C3 colony in order to prepare a glycerol stock of the strain.

6/4/2012	- Threw out old, incomplete inoculations from Friday. - Transformed I732005, R0010. - Miniprepped Venus and E0030 and put gDNA in the -20 temp box. - Did new inoculations on C0012, E0030, I732017, PSB1A3, PSB1AK3, PSB1K3. - Made a glycerol stock of the psB1C3 strain.
6/5/2012	- Made glycerol stocks of C0012, E0030, I732017, PSB1A3, PSB1AK3, PSB1K3 and put them in the -80 freezer. - Transformation of I732005 failed, so replated with rest of culture. - Transformed J04500 and PSB3T5. - Minipreped PUF Amp and protet CM20 (spilled 1 tube of this) and put gDNA in the -20 degree in the temp box. - Inoculated R0010.
6/7/2012	- Set goal to create RNA scaffold by 6/15, begun design.

6/11/2012	- Innoculated K12 - Transformed split GFP constructs BBa_K157005 and BBa_K157006 into DH5a cells - Constructed the first RNA scaffold draft - Inoculated both parts of split GFP, as well as J04500
6/12/2012	- Miniprepped K12 cells - RNA scaffold design will inculde two scaffolds which need to be stored into one plasmid and then ligated into separate plasmids - Made glycerol stock of both split GFP parts, J04500, and miniprepped both split GFP
6/13/2012	- Transformed Pertobrick into K12 cells - Constructed the first draft of the scaffold, but it needs review. - Second, unique scaffold needs to be designed
6/14/2012	- Plated Petrobrick transformations; inoculated cell growth using CM resistance - Finalized an updated version of the scaffold based on new data of PUF binding. Final modifications need to be made before sent for synthesis. - Performed PCR: (2) 50 uL tubes of control (no PUF binding site) and (2) 50 uL tubes with the PUF binding site. Used WT PUF as a template and phusion as the polymerase. Ran gel and saw a result of all primer dimers.
6/15/2012	- Made plan to digest cells in order to see whether or not petrobrick was transformed into K12. - Final modifications made on the scaffold design. The construct is ready for synthesis. - Second try of PCR: Did a temperature gradient of 55-63-71 degrees Celsius. Used the WT PUF as template DNA. Gel results show only primer dimers, the faintest of faint lines at around 700 base pairs, but it's not a positive result. - Miniprepped pTET is in the "temporary" box of the -20 degree Celsius freezer.

Team:UIUC-Illinois/Notebook/LabNotebook

From 2012.igem.org

Revision as of 02:31, 4 October 2012

Lab Notebook

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