Martin+K.+(RP+Summ+14)

Martin - ok good job on the lab work. Your Wiki could use some pics and also put the weeks in Reverse Chronological order. - Dr. B 072114

Martin - add in Week 5 results and analysis and then put the Weeks in reverse chronological order (i.e. Week 5 on top). - You also want this to be more of a summary of results and less focus on the procedural steps.-- DR. B

Week 8

7/21 Today we ran PCR to compare how different pfu polymerase performed in comparison to one another. Afterwards we made an oligomix using 38 different primers.

Unfortunately our gel was not made right, it was made with water instead of TAE buffer...

7/22 Today we ran another PCR, same as yesterday, with the correct gel. To our disappointment, we found out that our pfu polymerase was degraded/ denatured, and we got no bands on our gel whatsoever.

7/23

As a learning experience we made a PAGE gel with the extensive help of Zain.

7/24 We made another PAGE gel and ran it, because we messed up the first one.

WEEK 7

7/14

We ran a gel comparing pgbr 22 and pfu polyermase in a gel today, and also did our first PCR.

It ran decently, however our control lane had contamination, and the DNA production leveled off in the 30 ng lane, as seen in the intensities of the bands.

7/15 We ran another transformation efficiency using our calcium competent cells we spun down from week 6. 3 plates, left them overnight.

7/16

Today we ran another 2 PCRs testing. 1 PCR used taq like the first PCR but the other used our pfu polymerase as the enzyme.

We also ran gels to test them.

7/17

Based on the PCR's we ran yesterday, we decided to run temperature gradients altering the annealing temperatures to see which ones would work best.

7/18

Of the 8 PCR samples from yesterday, 2 of them worked best. We ran another PCR and gel based on these two temperatures.

WEEK 6

7/9 Today we continued to purify pfu polyermase, by running it in a centrifuge with JA 20 rotor.

7/10 The competent cell procedure! Stayed up to almost 7 repeatedly spinning down tubes with solution in JA10's and JA 20's. Almost 4 cycles.

7/11 Today we visited the J. J. Pickle research campus, and its giant super computing center!

We saw the Stampede system and got to look at it up close.

WEEK 5

7/1 Today we began to purify cloned pfu polymerase. We started by growing overnight cultures of LB and ampicillin and incubated them. (put them in incubator, NOT incubate them with our bodies)

7/2

We ran a gel of pGBR22 in a restriction enzyme digest. We used EcoRI and pvuII, and then with both.

7/3 We sonicated and centrifuged cells in order to isolate pGBR22.


 * WEEK 4**

COMPUTER LAB DAY

To start off the week, we copied the nucleotide sequence for pGBR22 onto a text file.Not all the bases were read/ sequenced, so we cut the code several hundred bases.

We then took the remaining code and did a BLAST search, which compared our sequence to known sequences of other organisms to look for similarities in the code. We discovered that the pGBR22 plasmid originated from a coral species.

The DNA sequence we found coded for a protein, so we translated the code we had into sequences of amino acids.

Finally, we took the pGEMT sequence and took the reverse complement, which we inserted into the original code that we found.Afterwards we did a virtual cut with restriction enzymes, and virtual gels.

MIDI PREP

The pGBR22 plasmid we grew in DH5alpha now replicated plenty of times. However, we now had to extract our DNA from the cells themselves, while separating the rest of the cell stuff.

We took the cells in the broth we had grown, and centrifuged them for 20 minutes, which settled the DNA at the bottom of the tube.

We then removed the LB broth in the tube, leaving just the purple plasmid DNA.

The midi prep procedure then began, which involved inserting lots of various buffers (P1,P2,P3) and filtering of solids through syringes and a QIAfilter and QIAprecipitators. What remained was then transferred into a 1.5 mL tube.

We nanodropped our extracted plasmid to get our concentrations. (163.8 ng/uL)

6/25

We took our grown and purified plasmid off to sequencing in the ICMB core facilities. Afterwards, we prepared leveral Agar plates with LB media. We added ampicillin to one plate and kanymycin to the other plate.

6/26-PLOT TWIST!

Unfortunately, we left out our midprepped plasmids on ice bucket overnight, which melted and caused partial degradation of our DNA(possibly?). So we had to make a new batch of purified plasmid from the backup flask we had in the fridge.

more unfortunately, I lost the plasmid i was working with due to a spill while pipetting. So instead... I learned how to prepare agarose gels!

6/27

Today we ran more prepared gels on the midiprepped plasmids made yesterday, and also sent them off to sequencing..

Week 3
For our first day in the lab, we engaged in a short exercise on accuracy and precision, involving pipetting various volumes of water!

We used 3 types of pipettors, each attuned to a different volume of water
 * P1000, P200, and P20.

With each pipettor we would pipette two different volumes of water. 850 uL and 300 uL with the P1000, 155 uL and 25 uL with the P200, 2.5 uL and 18.25 uL with the P20. We ran three trials with each volume and calculated average mass pipetted, standard deviation, volume, percent error, and coefficient of variation.

We then recorded our data in an excel spreadsheet and made four graphs displaying the average mass, standard deviation, percent error, and coefficient of variation.

SOLUTIONS AND BUFFERS!!!! (Day 2)

On day 2, we made lots and lots of solutions and buffers, with the help of a mentor. (forgot name??)

We calculated amounts needed to make solutions and buffers at specified volumes/concentrations.

We made LB broth, Tris base, NaOH, Tris SDS buffer, and 30% ethanol.

NANODROPS AND DNA SEQUENCING (Day 3)

On day 3, we finished with solutions and buffers, and learned how to utilize the nanodrop machine. We used nanodropping to determine concentrations of DNA in a solution, in this case pGBR22. We used this information to calculate the volume of plasmid that we would pipette into a centrifuge tube, along with nanopure water to create a 11uL solution that we sent off to a DNA sequencing facility,

GROWING CELLS IN SOLUTIONS (Day 4)

Starting on day 4, we began to grow the pGBR22 plasmid in DH5 alpha cells. We grew 3 plates each with different concentrations of plasmid, we grew the cells overnight in the incubator at 37 degrees Celsius.