Difference between revisions of "Team:Carnegie Mellon/Notebook"

Goal: To do Miniprep to purify the DNA. It is a small scale isolation of plasmid DNA from bacteria. Then do restriction enzyme digest on plasmid DNA and gel electrophoresis. This technique can be used for cleaving DNA molecules at specific sites, ensuring that all DNA fragments that contain a particular sequence have the same size; furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment. The cleavage method makes use of enzymes isolated from bacteria called restriction enzymes. They are able to cleave DNA molecules at positions at which particular short sequences of bases are present.

• Did MiniPreps of MACH cells expressing fluorescence to isolate plasmid DNA from MACH cells (protocol here)
• Performed restriction enzyme digestion on J23108, J23109, J23111 constructs
  For promoters, PST1 & Spe1 were used. For insert, xba1 and Pst1 were used.
• Ran an Agarose Gel Electrophoresis
• Cut digests from gels

For next time: Will prepare Colony PCR to determine if we got the correct restriction sites and sequences from the given interlab devices.

Goal: To submit minipreps for sequencing, will get results in two days and compare our constructions.

• Did minipreps on samples where the devices were incorporated in (J promoters & I3504)<.li>
• Send minipreps off for sequencing.
  5 µL water, 5 µL sample, 1 µL of each primer

Goal: Make competent MACH cells.

• Made competitent MACH cells (protocol here)
• measured fluorescence of GFP for 6 samples
  108A, 108C, 109A, 109C, 111A, 111B

For next time: Since we did it without MACH cells, will redo tomorrow with MACH cells and biosensor to compare results.

Goal: Observe Sequence Data and compare to our own plasmids. Redo TECAN results with blank and biosensor to see differences in fluorescent data.

• Compared sequence data to registry/our own plasmids. Figured out that no promoters got inserted in. Decided to send in Minipreps of the parts of the original devices for sequencing to see if the given parts were what we expected.
• Redid fluorescence reading
  Very evident difference, shows the presence of GFP

  May redo in the future in order to do the trianalysis.

Goal: Streaked out plates, continue competent cell protocol.

• Streaked out MACH cells, Top10, and Biosensor Estrogen - on to LB plates. The biosensor estrogen negative plates had kanamyacin antibiotic on it (10 ul + 50 ul water).
• Began TOP10 Competent cell protocol (protocol here).
  Diluted TOP10 overnight culture 1:100 for a 2ml volume in LB. Grew it from 10a-5p. Then added 300 ul to each 500 ml of LB in sterile 2L flasks. Placed in 18C shaker overnight after removing the magnetic stir rod.

Goal: Get back plasmid sequences to determine what went wrong with what we did. Restreaked the plates and competent cells growth is slow therefore will continue procedure Monday 6/8/15.

• Placed growing TOP10 cells in 2 L flasks in cold room because it was taking too long to grow in 18C shaker. Will place back in shaker Sunday night so that it can be ready on Monday to use (need OD600 of 0.4-0.6.)
• Further sequencing results on original devices sent out on 6/3/15 were obtained on 6/5/15. It showed that the promoters have 2 additional Spe1 cut sites which meant that they were cut out, which is why none of the transformants had the promoter in them.
  We notified iGEM, and they will thank us on their website for finding their mistake.
• Started Transformations for New Set Today.

Goal: Set up culture preps for minipreps

• Set up culture preps for minipreps (5ml) with 5ul of chlorophenical.
  Samples: 106A, 101A, 101B, 101C, 101D, 117A, 117B, 117C, 117D

  Left overnight in 37C shaker.

Goal: Redo the interlab study with the new devices given.

• Forgot to do ligation and transformation, therefore we will redo this tomorrow.

Goal: Redo digestion of the new transformations and determine if they fluoresce.

• Redid the Digestion because we forgot to do transformations.
• Did 3504 digestion with 101B, 106A, 117C
  For promoter: 10 µl of MiniPrep, 4 µl of H20, 2 µl of green buffer, 2 µl of EACH enzyme (PST and SPE1)

  For insert: 12 µl of MiniPrep, 2 µl of H20, 2 µl of green buffer, 2 µl of EACH enzyme (PST and Xba1)
• Put digests into a gel & washed gel with 1:1 buffer and put it into 42C heat shock for ~30 minutes
• Did PCR purification
• Ligation done using 4 µl of promoter, 4 µl of insert, 1 µl of buffer (t4 ligase buffer) LIG, 1 µl of T4 ligase
• Did transformation for lacYFP, FFluc, PRE, POST (protocol here).
  For lacYFP (can plate): 35 µl of cells, 1 µl YFP

  For FFLuc, PRE, POST (5mL LB AMP cultures): 35 µl of competent cells, 1 µl of such samples

  For our samples: 50 µl of competent cells, 10 µl of our mix (3504-101B, 106A, 117C)
• Also redid competent cells (autoclaved the 500mL LB, store TOP10 into 37C put in around 1microliter ← slow growth so put in more in order to grow faster)

For next time: See if our devices are green fluorescing tomorrow and to fixate our cells to the W strain in the competent cells being made.

Goal: Screen the transformants for ones that uptook the plasmid and to check if competent cell culture is ready.

• Screened the plates with transformants using UV light.
  Screened the plates with transformants using UV light.
• Set up 5ml culture using one colony from each (106A, 101B, 117C) for miniprep. Grew overnight in 37C shaker. Picked colony based on fluorescence using the UV light.
• Competent cells still did not reach the appropriate OD (currently 0.1 at 9:30a). Allowing it to shake overnight again.

For next time: Purify and isolate the transformant DNA using miniprep on the cultures that were set up today.

Goal: Purify and isolate the transformant DNA using miniprep on the cultures and then send it out for sequencing. Begin the competent cell protocol.

• Did miniprep on the 5ml cultures containing colonies of 101B (J23101 + I31504), 106A (J23106 + I31504), and 117C (J23117 + I31504)
• Sent out the DNA for sequencing.
• Competent cell culture was still did not have an OD of 0.4-0.6. Will check again tomorrow morning.

For next time: Begin PCR amplification on Gaussia Luciferase using Phusion high fidelity DNA polymerase.

Goal: Finish competent cell protocol and start the Gaussia Luciferase experimentation.

• Competent cell culture was within the OD. One of the competent cell cultures were within the appropriate range (at OD of 0.48). The other one wasn’t (at OD of ~0.2). We just started the competent cell protocol on both cultures but kept them separate so that we could compare their efficiency. The “Making competent MACH T1 cells” protocol was used for making competent TOP10 cells (protocol here).

Goal: Verifying the sequences obtained to the constructs that were made. Check the competence of our competent cells.

• Verified by comparing the sequence of our constructs to the sequence got from the samples we sent out.
  They seemed to be accurate with a few changes.
• Checked competence of competent cells using the iGEM competent transformation efficiency kit.
  Tubes labeled 1 w/ X conc are from the culture with OD within the range of 0.4-0.6. Tubes labeled 2 w/ conc are from culture that did not have an OD within the range of 0.4-0.6.

  Tube 1 with concentration 0.5, 5, 10, 20, and 50 picogram (5 samples in total)

  Tube 2 with concentrations 0.5, 5, 10, 20, and 50 picogram (5 samples in total)

  Tube with MACH cells.

For next time: Will count the number of colonies and determine the transformation efficiency of the cells.

Goal: Verifying the sequences obtained to the constructs that were made. Check the competence of our competent cells.

• Found nothing in both our competent cell plates and MACH plates from yesterday, using the DNA iGEM transformation efficiency kit.
• Redid Competent cells and MACH cells with plasmid DNA
• Plated both 400 µl and 40 µl for each competent cell transformation (total: 6 plates) & put in 37 °C incubator.

For next time: Check if our competent cells worked.

Goal: Verifying if our competent cells worked since it didn’t work using the DNA from iGEM.

• Checked the plates and found cells in both MACH and competent cells.