From 2014.igem.org
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| - | <th colspan="2" scope="col">Plasmids from addgene:<br> Genes: Bm3RI, BetI, Ph1F, SrpR, LrmA <br> | + | <th colspan="2" scope="col">Decided to make the following Level 0 Coding Sequences:<br> C0040_CI <br> |
| - | Terminators: L3S2P21, ECK120015170<br> Destination Vectors: pICH89921, pICH82113 | + | C0080_CI <br> E0040_ID <br> E0030_ID |
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| - | <ul><li>Struck out genes and terminators onto Amp plates <li>Struck out Destination Vectors onto Kan plates<li>Picked two colonies from each plate- one for minipreps & one for frozen stocks <li>Ordered primers for genes and terminators <li>Miniprepped terminators and genes <li>Made frozen stocks for all plasmids <li>Diluted primers and did PCR reactions for genes and terminators </ul> | + | <ul><li>Used Phusion PCR to make the parts listed above and then, performed PCR cleanup to purify the DNA fragments. |
| | + | <li> Upon quantifying all clean-ups, I found that the concentration for the E0040_ID (4.4 ng/uL) cleanup was lower than that of the negative control (6.1 ng/uL). So, I repeated the Phusion PCR for that part and ended up with a concentration of 31.9 ng/uL. |
| | + | <li> Performed MoClo Level 0 reaction to insert the purified constructs into backbones with a Cam resistant gene. |
| | + | <li> Transformed the MoClo plasmids on Cam plates to perform Blue-White screening and pick colonies that had successful digestion-ligation |
| | + | <li> Transformation didn't work because I used faulty Bioline cells. So, I repeated transformations using DH5a cells. |
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| | + | <li> Screened colonies further by performing Colony PCRs and running a gel. |
| | + | </ul> |
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Revision as of 22:03, 18 July 2014
Notebook: Fusion Proteins
| Notebook Overview |
June |
Week of June 23 |
Decided to make the following Level 0 Coding Sequences:
C0040_CI
C0080_CI
E0040_ID
E0030_ID
- Used Phusion PCR to make the parts listed above and then, performed PCR cleanup to purify the DNA fragments.
- Upon quantifying all clean-ups, I found that the concentration for the E0040_ID (4.4 ng/uL) cleanup was lower than that of the negative control (6.1 ng/uL). So, I repeated the Phusion PCR for that part and ended up with a concentration of 31.9 ng/uL.
- Performed MoClo Level 0 reaction to insert the purified constructs into backbones with a Cam resistant gene.
- Transformed the MoClo plasmids on Cam plates to perform Blue-White screening and pick colonies that had successful digestion-ligation
- Transformation didn't work because I used faulty Bioline cells. So, I repeated transformations using DH5a cells.
- Screened colonies further by performing Colony PCRs and running a gel.
Poured LB, LB+Amp, and LB+Kan plates
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Week of June 30 |
BM3RI, BetI, Ph1F, SrpR, LmrA, L3S2P21, ECK120015170
- Ran gel for previous PCR reactions
- Two terminators were too small to see well on gel
- PCR in triplicate for all genes and terminators as well as negative control
- PCR clean-up
- Didn't work for ECK120015170 because it was too small & fell through the filter
- Level 0 MoClo reactions for BM3RI, BetI, SrpR, LmrA, L3S2P21
- Redid PCR for ECK120015170
- Level 0 MoClo reaction for ECK120015170
- Transformations for all Level 0 MoClo reactions
- None of the transformations worked
- Accidentally used Pro cell strain with Cam resistance
- Redid MoClo reactions and transformations for all parts
- Ordered promoters for pBad, pTet, pA1LacO, and R0051
- Used colony PCR to verify transformations worked
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July |
Week of July 7 |
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This week I used the google glass for all protocols to give Wellesley College feedback on one of their software projects.
BM3RI, BetI, Ph1F, SrpR, LmrA, L3S2P21, ECK120015170
- Ran gel for colony PCR reactions
- Picked colonies to grow overnight for the parts that turned out well on the gel
- BM3RI, BetI, SrpR, LmrA, ECK120015170
- Redid transformations for Ph1F, L3S2P21, SrpR, and ECK120015170
- SrpR and ECK120015170 looked questionable on the gel so I redid them just in case
- Miniprepped overnight cultures and sent for sequencing
- Analyzed sequencing results and confirmed BM3R1, BetI, LmrA, and ECK120015170
- SrpR came back as LacZ, which means it wasn't properly transformed
- Picked confirmed colonies from stab plate, grew overnight, and made frozen stocks
- Miniprepped SrpR, Ph1F, and L3S2P21 and sent for sequencing
- Analyzed sequencing results and confirmed SrpR and L3S2P21
- Redid colony PCR for Ph1F because it had too low concentration for sequencing
pBad, pTet, pA1LacO, R0051
- Diluted pBad, pTet, pA1LacO, and R0051 primers
- PCR for pBad, pTet, and pA1LacO
- Held off temporarily on R0051 because we didn't have the R0051_Rev_B primer that we thought we had
- For each part we made AK and KB fusion site using the new fusion site, K, that we designed (ATGC)
- Level 0 MoClo reactions in DVL0_AB
- pTet-pBad, pTet-pA1LacO, pBad-pTet, pBad-pA1LacO, pA1LacO-pBad, pA1LacO-pTet
- Transformations for level 0 tandem promoter MoClo reactions in bioline cells
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Week of July 14 |
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In addition to my wetlab work this week I cleaned the lab, refilled stocks, and autoclaved backup supplies. I also talked to female high school students about iGEM and my experience with science and research to encourage them to pursue a science related field in university.
pBad, pTet, pA1LacO
- Created stab plate and picked two colonies from each transformation plate
- Miniprepped overnight cultures and sent level 0 tandem promoter MoClo parts in for sequencing
- Analyzed sequences
- Only the pTet-pBad tandem promoter turned out correctly
- Noticed that the pA1LacO promoter has a large repeating sequence
- PCR temperature that I used was too high (too specific causing reverse primer to bind to the wrong part)
- Redid PCR for pBad (AK) and pA1LacO (AK, KB)
R0051
- Received and diluted R0051_Rev_B primer
L3S2P21, SrpR
- Made frozen stocks from the confirmed colonies
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