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

FALL 2014
Nice work Carolyn - great having you in the lab group this year! - DR. B 121214

**WEEK 15**

 * Turned in final lab report, cleaned out personal box, and updated lab notebook.**

**WEEK 14**
Analysis FPLC: The peak on the FPLC graph of Pv6PG was around 53kDa which is likely to be Pv6PG. The tubes collected were 31-34 to avoid obtaining anything that isn't Pv6PG. This sample was nanodropped to find the concentration and then spun down in a concentrating tube to 1ml and snap-freezed using liquid nitrogen to be stored in the -80 degree Celsius freezer. 12/03/14 Purification of Pv6PG

=12042012- Fantastic work, keep it up!= =WEEK 13=



Figure 1. BL21(DE3) transformed competent cells with Pv6PG. Left: 50uL of BL21(DE3), 1uL Pv6PG plasmid and 200uL SOC media plated on LB agar + kanamycin plates after overnight incubation. Right: 50uL of BL21(DE3), 1uL Pv6PG plasmid and 50uL SOC media mixture plated on LB agar + kanamycin plates after overnight incubation. 11/18/14

=**WEEK 12**=

11/11/14 Making LB 4 liters of LB made for future large scale expressions. 2 expressions (each 2L) will be carried out side by side with one acting as a backup in case FPLC does not work out

__**For 2L LB**__ - 20g Bacto-tryptone - 10g Bacto-yeast extract - 20g NaCl

- Combine in 4L flask with 2000 mL of dH2O - Stir until dissolve completely with stir bar - Autoclaved

=WEEK 11= FPLC 11/4/2014: Purification of Pv6PG. Samples 2-6 were stored in the 4 degree Celsius fridge.



WEEK 10



 * WEEK 7**

Analysis: Figures 4 and 5 show that the forward and reverse primers might not have been fully attached which left a lot of small pieces of DNA to run down the gel.
 * WEEK 6**


 * WEEK 5**

Analysis: The contamination shown in Figure 3 may have been caused by removing the comb out of the gel before it was completely dry. Lanes 1-5 were not usable.

Analysis: The gel was blank because instead of using EtBR, 0.5uL of red juice from a green PCR tube was used. 09242014- Good job, but don't forget to give an analysis and leave captions After two gels my secondary PCR has not worked. I think I need to go back and redo my oligo mix. Although it seems primary PCR worked in Figure 1 no bands for both the ladder and the sample appeared under the UV light. This means that my sample doesn't actually work, or my gel was not good because the ladder didn't show up as well.

Week 7
7/17/14 PCR Cleanup 07/17/14 PCR Squared ^*Runs 24-31

Week 6
Keep up the good work! You'll get it soon (: Which primers were wrong? Include a virtual gel of RE Digest to #|verify. -Grace 7/15/14 ___ PCR for pNIC-Bsa4 has not worked after three times. A fourth time will be attempted later using different #|primers. Results for RE digest are good but there are slight bands in lanes 5 & 6 under the 125ng band possible due to contamination?
 * Smears are okay for primary PCR because the primers had not been added to the bands did not separate

Oligo Mix
Done on July 2nd. 1 uL sample from each of the 36 wells were placed in a micro centrifuge tube with 64 uL of autoclaved nanopure water and stored in my box in the -20 degree Celsius fridge. This mix will be used in primary PCR for our target.

**FPLC YopH BL21**
7/3/14 - We concentrated the 33 kDa and 44 kDa samples to 1 ml. 0.5 ml of each of the samples were placed in the -80 degree Celsius freezer and the other 0.5 ml is in the -20 degree Celsius fridge.

Protein Expression
Pellet weight: 3.7g


 * Midi Prep Results **
 * Nanodrop**
 * Midi Prep DNA Sequencing **

Week 1 and 2