Chris+T.+(RP+Summ+14)

Fall 2014:
Week 12: PCR cleanup seemed to have failed since I could not find a concentration of my protein from Overlap PCR. I ran a PCR gel on the sample that I used to nanodrop and it turns out that my sample was lost in the waste during PCR cleanup. In order to fix this, I need to rerun another PCR squared since I used it all up for PCR cleanup. By rerunning a PCR squared I may see less contamination since it was my first time I performed both PCR squared and cleanup.





Week 11: Although contamination can be seen, there are distinct bands that match the 1122 bp size of my target Mt. D-ala-D-ala Ligase so I can proceed onward to PCR cleanup.

Week 10 1162014- Congrats on getting your secondary PCR to work, dont forget to distinguish which weeks are which

10/24/14

ALL HAIL THE HELIX FOSSIL!!! Secondary PCR finally worked! WOOT! Time to catch back up to Charina! The reason for my secondary PCR is working is probably because of the increase in annealing time by two seconds (12 seconds for annealing step).



Figure 2- Secondary PCR for Mt D-ala; D-ala. Inside lane 2 is 5 microliters of 1 kb DNA ladder and inside lane 4 is 12 microliters of secondary PCR sample.

Week 9:

Figure 1- Secondary PCR for Mt D-ala; D-ala. Inside lane 2 is 5 microliters of 1 kb DNA ladder and inside lane 4 is 12 microliters of secondary PCR sample.

Week 8:

The reason why my Primary PCR has not been working is because my oligo mix needed to be remade since it was from the summer. The secondary PCRs that I have been running have been failing to show any distinct bands that corresponds to the size of my target, 1122 bp. The only reasons that can explain why my secondary PCR has not been working is that my tail primers have degraded and/or a temperature gradient is needed during the Overlap PCR step. Apart from the PCRs that I have done today, virtual screening for my target is currently underway.



09232014- Good job, but don't forget to give an analysis

Week 7
Chris - ok keep pshing on the primary PCR. Also, at the same time work on growing up and cutting some pNIC-Bsa4 for cloning. - Dr. B072114 7/16/14 After multiple attempts for primary PCR, I am redoing my oligo mix since the streaks seen in the gel do not match the size of my protein which is 1122 bp.

Keep trying! Give analysis on the results, and make it clear your progress. -Grace 7/15/14 P-Nic Bsa-4 gel
 * Lane 1**: 1kb DNA Ladder
 * Lane 2-3**: Hailey’s stuff
 * Lane 4**: Skip
 * Lane 5**: Sample A (Chris T.)
 * Lane 6**: Sample B (Chris T.)
 * Lane 7**: Sample C (Chris T.)
 * Lane 8**: Sample D (Chris T).

Based on the results of the p-NIC Bsa 4 gel, the results matched my expectations since sample B and C showed bands which had the highest concentration 1:100 and 1:10 respectively. Whereas sample A, the least concentration (1:1000) had no visible bands. Since Sample D is the control, there was suppose to be no visible bands shown in the gel.

** Lane 1: ** 1 kb DNA Ladder

 * Lane 2: ** Uncut DNA template
 * Lane 3: ** EcoRI digest (Hailey)
 * Lane 4: ** PvuII digest (Hailey)
 * Lane 5: ** EcoRI and PvuII digest (Hailey)
 * Lane 6: ** EcoRI Digest (Chris)
 * Lane 7: ** PvuII Digest (Chris)
 * Lane 8: ** EcoRI and PvuII Digest (Chris)
 * Lane 9: ** EcoRI Digest (Charina)
 * Lane 10: ** PvuII Digest (Charina)

Description :Lane 1 has 1 kb DNA laddr. Lane 2 has the sample of uncut pGBR22 DNA template. Lanes 3, 6, and 9 have the sample cut with EcoRI. Lanes 4,8, and 10 are cut with PvuII. Lanes 5 and 9 are cut with both EcoRI and PvuII. Cuts with EcoRI are seen by one band and cuts with Pvu are seen by two bands.

Ahh - no week 5 ? - Dr. B

Week 5
Primary PCR and pNIC-Bsa4 PCR failed T_T FPLC for YopH and obtained Oligo mix



Week 4
Chris - good. Did you do some protein work also? Any protein gel results? The DNA agarose gels look good. I think your PCR worked nicely (you don't see the increasing gradient, but the bands are sharp and well defined (and all at the same migration point on the gel). There seems to be contamination in Sample C - but it might just be 'multimer' bands from lots of DNA. There does seem to be 'contamination' in Sample D - but this is probably just some carry over that spilled from lane 4 into lane 5. For the pNIC-Bsa4 midiprep - you will need to do a BLAST to compare your DNA Sequencing read to the known sequence to confirm it is correct. The known pNIC-Bsa4 sequence is in the GDrive. Nice work overall. Dr B

Analyzed DNA sequencing in computer lab DNA sequencing results from the pNIC-Bsa 4 we used in Midi-prep.



Chris - good. Did you measure cell pellet weights for the YopH expressions?
Is the DNA Sequencing result below of pGBR22 for a sample you had Midi-prepped? Or is it the sample that we already had? If the former - then include a Nandrop image so that we can get an idea of yield. - Dr. B

Protein Expression: Large culture for FTHap and sonicated the sample after lysing.

Nanodrop Images of pNIC-Bsa 4 that we used in Midi-prep

Made LB+Kan plates and transform bacterial competent cells (Bl21) with YopH + pNIC BSA 4 plasmid for plasmid prep of pNIC- BSA 4_v2
This week some people did Midi Prep and PCR.

DNA Sequencing Results of pGBR-22 already in lab.

Week 1 and 2
Transformation Efficiency for Tubes A, B, and C is 1.9*10^4, 4.8*10^3, and 4.8*10^3 respectively. People did DNA sequencing and made LB 