Betty+H.


 * Week 8**
 * pNIC-Bsa4 transformed with RpFabG RE Digest with SacII and PvuII (7/23/13)**

Betty pNIC RE Digest with RpFabG elutions from miniprep of transformed pNIC (with RpFabG) Lane 1: empty Lane 2: 1 kb DNA ladder Lane 3: uncut pNIC-Bsa4 control Lane 4: elution 1 (cut with SacII and PvuII) Lane 5: elution 3 (cut with SacII and PvuII) Lane 6: elution 4 (cut with SacII and PvuII) Lane 7: pNIC (cut with SacII and PvuII) Lanes 8-10: empty

Betty pNIC-Bsa4 cut with BsaI-HF Lanes 1-3: empty Lane 4: 1 kb DNA ladder (here, I was unsure if I had enough to show up, so I assumed it wouldn’t and added another ladder) Lane 5: empty Lane 6: 1 kb DNA ladder Lane 7: cut pNIC-Bsa4 (cut with BsaI-HF) Lane 8-10: empty
 * Week 7**
 * pNIC-Bsa4 cut with BsaI-HF (7/19/13)**

This was my third time making more cut plasmid; I made more in order to continue further rounds of cloning because none were, so far, working out.


 * RpFabG PCR squared product after PCR Cleanup (7/18/13)**



The above two figures were my PCR squared product after nanodrop. They were made and cleaned 7/17, but nanodropped the morning of 7/18. The average concentration is 261.8 ng/uL.


 * Good job on these! - DR. B 071713**
 * pNIC-Bsa4 (cut with BsaI) Nano Drop after PCR Cleanup (7/17/13)**



The sample I nanodropped in figure 1 and 2 were created by cutting pNIC-Bsa4 in five different tubes, each of volume of 50 uL as the protocol states. The five were then combined after the water bath and 15uL was taken to run a gel. The remaining 235uL were cleaned with the PCR cleanup kit and the results are shown above.


 * Week 6**
 * pNIC-Bsa4 (cut with BsaI) Nano Drop after PCR Cleanup (7/12/13)**



The sample I nanodropped in figure 1 and 2 were created by cutting pNIC-Bsa4 in four different tubes, each of volume of 50 uL as the protocol states. The four were then combined after the water bath and 20uL was taken to run a gel. The remaining 180uL were cleaned with the PCR cleanup kit and the results are shown above.


 * pNIC-Bsa4 (cut with BsaI) Nano Drop after PCR Cleanup (7/12/13)**
 * Figure 1.** The first nanodrop of my cut pNIC-Bsa4 after PCR cleanup.


 * Figure 2.** The second nanodrop of my cut pNIC-Bsa4 after PCR cleanup.

Overall, the concentration of my sample was low, but the 260/280 value of 1.8 represents the ideal purity in relation to the protein. The 260/230 value, however, was 1.1, lower than the desired 2.1 by nearly half.

Betty RE Digest Lane 1-2: empty Lane 3: 1 kb DNA ladder Lane 4: pNIC-Bsa4 (cut with BsaI-HF) Lane 5-10: empty
 * pNIC-Bsa4 RE Digest (7/11/13)**

I did a gel of pNIC-Bsa4 and cut it with BsaI to ensure that it would work. It cut twice, as it should, and the various sizes were appropriate.


 * pNIC-Bsa4 Midi Prep Nano Drop (7/10/13)**
 * Figure 1.** The first nanodrop of my pNIC-Bsa4.


 * Figure 2.** The second nanodrop of my pNIC-Bsa4.

I grew up pNIC-Bsa4, in 160mL LB Media with 160 uL of Kan, overnight on Monday and spun it down the day after. Today, I took the pellets and completed Midi Prep on it, the nanodropped the plasmid to determine the concentration. The average 260/280 value is 1.91 which is close to the desired 1.8, signifying a fairly high purity in relation to the protein. The 260/230 value, however, was 2.57, because it was fairly high in one nanodrop but a lot closer to 2.1 in the second.


 * PCR Squared (RpFabG Gene) Nano Drop after PCR Cleanup (7/09/13)**
 * Figure 1.** The first nanodrop of the PCR cleanup results.


 * Figure 2.** The second nanodrop of the PCR cleanup results.

The following are the nanodrop measurements for the PCR cleanup of Ricksettsia prowazekii FabG. The average concentration is 128.8 ng/uL, and the 260/280 average of 1.86 is very close to the desired 1.8 and the 260/230 average of 2.23 is very close to the desired 2.1.


 * Week 5**
 * Primary and Secondary PCR Gel (7/05/13)**

Lane 1: empty Lane 2: 1 kb DNA ladder Lane 3: primary PCR Lane 4: secondary PCR Lane 5-10: empty

My primary PCR showed up like a smear as was desired, and my secondary PCR showed up at a band at between 100 and 200 base pairs.

Betty pLIC for pNIC-Bsa4 Lane 1: empty Lane 2: 100 bp DNA ladder Lane 3: sample A Lane 4: sample B Lane 5: sample C Lane 6: sample D (negative control) Lanes 7-10: empty
 * PCR for pNIC-Bsa4 with pLIC (7/05/13)**

While this PCR succeeded in that lane 5 (sample C) has the most DNA, my control evidently had some contamination.

Betty Lane 1: 100 bp DNA ladder Lane 2: sample A Lane 3: sample B Lane 4: sample C Lane 5: sample D (negative control)` Lanes 6-10: empty
 * PCR Green for pGFP (7/03/13)**

The GREEN PCR proved relatively successful, with equally sized bands and minimal contamination.

__ Forward Primer: __
 * Virtual Plasmid for Rickettsia Prowazekii FabG in pNIC-Bsa4 (7/01/13)**

 5’ TACTTCCAATCCATGATTGACCTCACGGGCAAAAC 3’ 35 bp GC Content 45.7% 0 mM Mg2+ Tm 64.4 oC 1.5 mM Mg2+ Tm 71.7 oC 2 mM Mg2+ Tm 72.1 oC 4 mM Mg2+ Tm 73.1 oC 6 mM Mg2+ Tm 73.5 oC

__ Reverse Primer __ :

 5’ ACGGTGGTATGCTGATGGTTTAACAGTAAAGGTGGATA 3’


 * Reverse complement it: **

 5’ TATCCACCTTTACTGTTAAACCATCAGCATACCACCGT 3’ 38 bp GC Content 42.1% 0 mM Mg2+ Tm 64.1 oC 1.5 mM Mg2+ Tm 71.7 oC 2 mM Mg2+ Tm 72.2 oC 4 mM Mg2+ Tm 73.1 oC 6 mM Mg2+ Tm 73.5 oC


 * Week 4**
 * Protein Purification NanoDrop (6/27/13)**
 * Figure 1.** This nanodrop shows the results for Elution 1 of PfDXR.


 * Figure 2.** This shows the results for Elution 2 of PfDXR.

BHLK REdigest pGBR22 62513 Lane 1, 5-7: Betty Lane 8-10: Laraib
 * RE Digest Gel (6/26/13)**

Lane 1: sample A-EcoRI (with NEBuffer 2)Lane 2: 1kb DNA ladder Lane 3: uncut plasmid Lane 4: skipped Lane 5: sample B-PvuIILane 6: sample C-EcoRI+PvuII Lane 7: sample D-EcoRI (with NEBuffer for EcoRI) Lane 8: sample A-EcoRI (with NEBuffer 2)Lane 9: sample B-PvuII Lane 10: sample C-EcoRI+PvuII

The RE Digest showed relatively good results, though was not completely correct.

I did Midi Prep on 6/19/13 and submitted a diluted 12 microliter sample of my pGBR22 plasmid to the DNA sequencing lab that same day. The sequencing was completed within two or three days, but I did not run the sequence through the BLAST database until Monday the 24th. The results were good, however: 100% Montipora efflorescens GFP-like chromoprotein.
 * Midi Prep DNA Sequencing Results (6/24/13)**

BHLKpBGR2262013 Lanes 1-5: Betty Lanes 6-10: Laraib Lane 1: 100 bp ladder Lane 2: sample A Lane 3: sample B Lane 4: sample C Lane 5: sample D (negative control) Lane 6: 100 bp ladder Lane 7: sample A Lane 8: sample B Lane 9: sample C Lane 10: sample D (negative control)
 * Week 3**
 * Agarose Gel (6/20/13)**

The third time doing the pGBR22 PCR produced much better results than the previous two times.


 * DNA Sequencing (6/18/13)**

I submitted my order of pGBR22 plasmid on 6/11/13 to the lab and received my results within a couple of days. However, I didn't check it against the BLAST database until today. The results showed that my sample was correctly matched at 100% to Montipora efflorescens GFP-like chromoprotein mRNA.

BHLKVDS61813pGBR22 Lanes 1-5: Betty Lanes 6-10: Laraib Lane 1: 1 kb ladder Lane 2: sample A Lane 3: sample B Lane 4: sample C Lane 5: sample D (negative control) Lane 6: 1 kb ladder Lane 7: sample A Lane 8: sample B Lane 9: sample C Lane 10: sample D (negative control)
 * Agarose Gel (6/18/13)**

The bands showed up a lot better for my samples of pGBR22, but I proceeded to redo the PCR again anyway.


 * Week 2 - Betty great job. You will list your Weeks in reverse chronological order (you stared on Week 2 technically) . Thanks :) -- Dr. B**
 * Transformation Efficiency (6/10/13)**
 * Figure 1. ** Plate A, which contained 1ng of pGFP plasmid with DH5alpha bacteria on LB+Amp Agar plates. Plate A showed no growth at all, demonstrating either an efficiency of zero or suggesting that something was done incorrectly or went wrong.


 * Figure 2.** Plate B, which contained 5ng of pGFP plasmid with DH5alpha bacteria on LB+Amp Agar plates. Plate B showed some growth, although only in a singular spot on the plate, suggesting that this plate was either done incorrectly or something else went wrong to show lack of growth.


 * something happened to the picture, so I have to reupload it from my email**
 * Figure 3.** Place C had the most colonies. It contained 25ng of pGFP plasmid with DH5alpha bacteria on LB+Amp Agar plates.

The numbers from the three plates were not similar, which could be expected due to the varying amounts of plasmid that were applied to each plate.

BHLKPCRpGBR22061313 First four lanes: Betty Last four lanes: Laraib Lane 1: 1 kb DNA ladder Lane 2: sample A Lane 3: sample B Lane 4: sample C Lane 5: sample D (negative control) Lane 6: 1 kb DNA ladder Lane 7: sample A Lane 8: sample B Lane 9: sample C Lane 10: sample D (negative control)
 * Agarose Gel of PCR Plasmid (6/13/13)**

My four lanes showed no bars except for lane 3. In lane 5, no image would be expected, as it contained no DNA. However, lanes 2, 3, and 4 would have been expected to contain increasing amounts of visible bars, demonstrating amplification, as lane 2 contained the least amount of DNA and lane 4 contained the most.