Team:Caltech/Notebook/Coliroid

Coliroid Notebook

6/22/12

Transformed M30109 and R0082, M30109 had 3 colonies and R0082 had none.

Re-transformed R0082, but with no colonies.

Ran PCR of R0082 with OneTaq polymerase, gel had no band.

PCR R0082 with vf2 and vr primers and Phusion polymerase. Gel had confirmed band length. PCR purified and had 34.6 ng/ul.

Both R0082 and positive control plates had colonies. Transformed RFP (E1010) and lacZ and plated.

PCR lacZ and pVH001 (mCherry-AAV Victoria's construct) and ran gel with correct band length for lacZ. There is lacZ DNA, but past lacZ transformations have not worked. Miniprepped overnight cultures of R0082 (43.6 ng/ul) and M30109 (28.9 ng/ul).

Transformed lacZ again and sent R0082 and M30109 for sequencing. Started overnights of R0082, E1010, and MG1655.

We transformed pSBC13 into Mg1655 on 3 different plates. We put 1 microliter of Mg1655 on 1 plate, 10 microliters on the second plate, and 100 microliters on the third plate, in order to figure out which one would make the best looking plate of red E. coli. We also miniprepped samples of R0028 and E1010.

We started another culture for M1655 since our previous culture was made with contaminated LB. Then we re-digested the R0082 and ran a gel comparing the digested R0082 with the undigested. The digest was successful, so tested the concentrations of DNA in the R0082, the E1010, and the lacZ samples with the Nanodrop. There was not a high enough concentration of the E1010 DNA, so we could not use it in ligation. We ligated the R0082 with the E1010 and plated the plasmid. Also, we ordered primers for mCherry-AAV, mCherry-LVA, and mCherry.
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7/9/12

We found that there was growth on the ligation plate and no growth on the negative control plate, which was good. We checked to make sure that the ligation of R0082 and E1010 worked by running a PCR of 6 different colonies on the ligation plate as well as a sample of the undigested R0082 and then running a gel of these things.

Started 5 cultures of ligated colony of RFP and R0082 to shake for 5-6 hours and then streak out on plates. Ran gel of lacZ PCR product.

Ran gel to check lacZ digest, and PCR purified. PCR mCherry-AAV/LVA and streaked plate of M30109.

Ran gel for mCherry, PCR purified mCherry, M30109 had no colonies. Started overnights for R0082.

To do: Digest mCherry and R0082, run gel to make sure. PCR purify, then ligate mCherry+R0082 and lacZ+R0082

Ligated R0082 with lacZ, mCherry-LVA, mCherry-AAV and plated. Transformed another lacZ part (BBa_I732019).

There was growth on the lacZ, mCherry-AAV, and mCherry-LVA ligation plates. We did a colony pcr and re-struck colonies for these plates as well as for the E1010-R0082 ligation plate.

We ran a gel on the colony pcrs of the lacZ, mCherry-AAV, mCherry-LVA, and E1010 ligation plates. The gel showed that the ligations had apparently not worked, so we re-digested the mCherries and the R0082 to start over the process of ligation.

We ran a gel on the digests of the mCherries and R0082, and it appeared to have worked, so we ligated mCherry-AAV + R0082 and mCherry-LVA + R0082, then ran a gel.

We re-did a ligation on the mCherries and R0082 because it appeared from the gel that the ligation from Friday did not work.

Ran a gel of re-ligated R0082 and mCherry-LVA,mCherry-AAV.

Plated the mCherry ligations 2, 3, and negative control.

The four mCherry ligations plates have colonies and the negative control plate has some, but not many compared to the ligated colonies. Colony PCR on the ligations and negative control. PCR mCherry-LVA and mCherry-AAV with dpn1 digest? Make plates with agar embedded.

Ran a gel and imaged the ligations and confirmed the ligations worked. Overnights of ligation for miniprep for tomorrow morning.

Started overnight cultures of mCherry ligations with varying salt concentrations (0, 0.25, 0.5, .75, 1 mol NaCl) and one growing in water. Sent mCherry ligations off for sequencing.

Overnight cultures have no apparent redness, looked at cultures with plate reader for any RFP, but there is very little which may be background. The RFP created may have been degraded by the degradation tags.

Amplify lacZ out of E. coli genome.

Transformed and plated multiple parts: cph8, cph8 + PCB (missing terminator), PCB from Lagarias, cph1 from Lagarias.

Every plate has many colonies, too many colonies.

The plates in the fridge were contaminated, and my plates had some contamination, so I need to throw them out and transform again! Made new batch of Carb-Amp, Chlor, and Kan plates.

Re-transformed all plates: K321224, K321227, PL-PCB, PTYB2-cph1, I12013.

PL-PCB, PTYB2-cph1, and I12013 had colonies. Restreak them out to isolate a colony and restreak M30109 from Agar Stab from parts registry.

Miniprep M30109 and send off for sequencing. Order lacZ primers. Double check mCherry constructs. Add terminator to E1010 + R0082 constructs.

Transform R0040 (tetR promoter).

PCR mCherry-LVA/AAV. Digest with E & S and ligate to E & S digested pSBC13. Transform mCherry-LVA/AAV, M30109, PCB. Make overnights of R0040. Order primers for regular mCherry construct. Order primers to PCR out cph8 from M30109.

Miniprep R0040 and send for sequencing. Digest, ligate and transform R0040 with mCherry-LVA/AAV.

Make overnights of R0040 + mCherry-LVA/AAV.

Miniprep R0040+mCherry-LVA/AAV.

Send R0040+mCherry-LVA/AAV for sequencing.