IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/25

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Team Vector
Last week we had made cultures of V9, V10, V11 and V12 vectors and stored pellets of the resulting cells over the weekend.

We miniprepped the cells to obtain the plasmids. We did so using the Qiaquick Miniprep kit. As an initial test that we had obtained the plasmid we expected we digested using PstI and HindIII and ran on a gel. The gel (shown below) showed the presence of the expected bands.

To verify that we had inserted the sequence that we expected into the plasmid we planned on sending them for sequencing by GeneWiz. GeneWiz require concentrations of 80ng/uL. Our nanodrop results showed that we had obtained low concentrations from our miniprep (<44ng/uL). To achieve higher concentrations of plasmid we re-picked colonies and incubated cultures for longer. We miniprepped the cultures and obtained higher concentrations of the plasmid.

We did not have a large enough volume of plasmid for sequencing so we transformed the miniprepped plasmid back into E. coli and grew on LB + Kanamycin. We picked colonies and grew cultures. We obtained the plasmid using a miniprep.

To create the insert for the V7 and V8 vectors we annealed oligos. We ligated the insert into the V7 and V8 backbones and transformed into E. coli. We incubated the E. coli overnight on LB + Kanamycin plates. Colonies grew on our negative control and colonies were unevenly distributed on other plates. This suggests that there may have been a problem with our plates or ligation so we should repeat those steps.

We did not have much O1 or O2 backbone remaining so we digested the V1 and V2 plasmids with SacII and SpeI to obtain more. We ran on a gel and gel purified the backbone.

Team Flavor
We began this week trying to ligate the pENTCUP2 promoter, NosT terminator, and NosT terminator plus stop codon into the V0120 vector. We ran into several problems during this process.

We had previously digested the three inserts as well as the promoter with XbaI and PstI fast digest enzymes, ligated, and transformed the ligated vectors into e. coli. Unfortunately, we did not find any colonies of cells after over 14 hours of incubation. Furthermore, on Friday June 18th, after digesting all three inserts and vector and running them on a gel, we did not see any NosT + stop DNA. Therefore, on Monday June 21, we began by digesting more NosT + stop and V0120 vector with xba1 and pstI fast digestive enzymes. The gel showed DNA length to be consistent with the given parts. (See gel below)


 * Gel Lanes (both inserts and vector were digested with xbaI/pstI): 1. 1kb plus ladder. 2. pENTCUP2 promoter. 3. NosT terminator. 4. NosT + stop terminator 5. V0120 digested.


 * Gel Lanes (both inserts and vector were digested with xbaI/pstI): 1. 1kb plus ladder. 2. NosT + stop terminator. 3. V0120 digested.

Upon gel extraction and gel purification, we ligated the inserts into the vectors with a 2:1 insert to vector ratio (previously we had been using a 4:1 insert to vector ratio). We then transformed the plasmids into e.coli and plated; no colony growth was seen the next morning.

We used Team Vector's R3 ligation (they have successfully ligated and transformed) as a positive control. However, the bacteria containing the ligated plasmids were plated on Ampicillin plates, which turned out to be the wrong resistance.

Troubleshooting
In order to get a better understanding of the problem with our ligation, we ran a diagnostic gel with the V0120 backbone and vectors B15 and B21.

Gel Lanes: 1. 1 kb plus ladder 2. v0120 undigested 3. v0120 eco/spe 4. v0120 eco/xba 5. v0120 spe/pstI 6. v0120 xba/pst 7. v0120 ecoRI 8. v0120 xba1 9. v0120 pstI 10. v0120 spe 11. blank 12. B15 (StrepII tag) xba/pstI 13. B21 (YFP) xba/pstI

We concluded from the gel that xba1 is the problem in our ligation step. The pattern of bands in the v0120 xba1/pstI digest along with the v0120 xba1 digest illustrate that xba1 is not cutting correctly.

We began a slow enzyme xba1/pstI digest on Friday, but the gel was run in mismatching buffers (TAE and TBE).


 * Gel Lanes (inserts and vector digested with xba1/pstI): 1. 1 kb plus ladder. 2. pENTCUP2 3. NosT 4. NosT & stop  5. v0120

Valencene
Digestion of PCR products aimed to isolate valencene gene showed very short length on gel. These are not the correct size and are most likely primer dimers. PCR products were digested with EcoRI/SpeI. 5 μL of each sample was digested with pstI to be sure that the pstI site was in the sequence of DNA.


 * Gel Lanes: 1. 1 kb plus ladder. 2. Flavedo EcoRI/SpeI.  3. Flavedo PstI  4. Open  5. Fruit EcoRI/SpeI.  6. Fruit pstI