Daren+N.+(Springer)

1162014- Keep up the good work
 * 120042014- Nice Work**

9232014- Great job, and nice images

Daren - put these in reverse chronological order also. - DR. B

Week 14 & 15 Figure 1: (12-5-14) 1kb ladder in well 1 with PCR Squared from the 61 degree Celsius secondary PCR sample in well 2, 3, 4, and 5.

Analysis: Shown in Figure 1, PCR squared was a success by the fact that there were distinct band just below the 1000 DNA sequence band from the ladder. Stp1 is 750 DNA sequence long, so the band are in the correct position. In addition, there is not much contamination of the PCR sample.

Conclusion: Now that PCR squared is successful, future steps in VDS is so proceed with PCR clean up even though the PCR squared sample is not as contaminated at it could have been.

Week 11, 12, 13
 * Figure 1: ( 11-04-14 1pm-5pm ): 1kb ladder on well 2 and from well 3-10 there is a temperature gradient from 50.1OC to 58.1OC for secondary PCR **[[image:dtn548_110414_AgaroseGelSecondaryPCRTargets.JPG width="400" height="306"]]
 * Figure 2: ( 11-04-14 5pm-8pm ) 1kb ladder, primary with 1ul Stacy’s oligo mix, primary with 2ul of Stacy’s oligo mix, and primary with 1ul of Q5 polymerase in well 3, 4, 5 and 6 respectively. **


 * Figure 3: ( 11-05-14 4pm-7pm ) 1kb ladder in well 2, and from well 3-10 there is a temperature gradient from 50OC to 58OC for primary PCR using Stacy’s oligo mix and the standard components for the sample in the primary PCR protocol **
 * Figure 4: ( 11-13-14 3:30pm-8pm ): 1kb ladder in well 3 with a primary PCR using a 2ul of new oligo mix and 1 ul of Q5 polymerase. **
 * Figure 5: ( 11-14-14 10am-1pm ): 1kb ladder in well 1, and from well 2-9 there is a temperature gradient from 58OC to 68OC for secondary PCR **

Analysis: Within Figure 3, the band was very faint and I had to alter the image in order to see it. To make sure that my secondary worked I ran the procedure again, but with a temperature gradient from 50.1OC to 58.1OC shown in Figure 4. As seen, there were no bands in any of the wells with the secondary PCR sample indicating that the experiment failed. This can be from the fact that my original primary PCR failed and that the smear is from the blue dye. Therefore, that secondary PCR in Figure 3 failed, and that the faint band could have been a leak from my partner’s sample. Due to the fact that my primary PCR did not work, I used Stacy’s oligo mix and altered the produce conveyed in Figure 5. The experiment failed shown by the lack of smears in well 4-6. I decided to do a temperature gradient from 50.1OC to 58. 1OC for the annealing cycle shown in Figure 6, but again the procedure failed. I approached this problem by starting over by making a new oligo mix, and used the standard protocol of primary PCR with the temperatures shown in orange. Shown in Figure 7, there is a definite smear in well 4, so the next step would be to move on to secondary PCR. For my secondary PCR, I used a temperature gradient once again from 58OC to 68OC for the annealing cycle. As shown in Figure 8, some of the wells were a success while some were not by either the bands or lack of bands.

Conclusion: Now that secondary PCR was a success the next step in VDS is to start PCR^2 to amplify even further to use to insert it into the pNIC-Bsa4 vector. Week 8,9,10 Secondary PCR (10-29-14 and 10-30-14)
 * Figure 1: (10-29-14): 1kb ladder in well 2, primary PCR in well 3, secondary PCR in well 4, primary PCR with 2ul of oligo mix in well 5, secondary PCR from the primary PCR with 2ul of oligo mix in well 6, primary PCR with 1ul of Q5 polymerase in well 7, secondary PCR from the primary PCR with 1ul of Q5 polymerase in well 8. Using regular temperature, and the times in orange in the primary PCR protocol. **


 * Figure 2: (10-30-14) 1kb ladder in well 2, secondary PCR from the primary PCR with 2ul of oligo mix in well 3, secondary PCR from the primary PCR with 1ul of Q5 polymerase in well 4. Using regular temperature, and the times in orange in the primary PCR protocol except added 2 seconds to the annealing cycle. [[image:dtn548_103014_AgaroseGelSecondaryPCRTargetsSECONDTRY2.JPG width="408" height="503"]] **
 * Figure 3: (10-30-14) 1kb ladder in well 2, secondary PCR from the primary PCR with 2ul of oligo mix in well 3, secondary PCR from the primary PCR with 1ul of Q5 polymerase in well 4. Using regular temperature, and the times in orange in the primary PCR protocol except made the elongation time to 45 seconds. **

Analysis: In Figure 1, I used all 3 samples from my primary for secondary PCR, but since there were no solid bands, the experiment failed. For Figure 2, I added two seconds to the annealing time to insure that the primers had enough time to bind to the DNA strand, but once again the experiment failed due to the lack of bands. For Figure 3, I upped the elongation time to 45 seconds so that the Q5 polymerase would have enough time to replicate the DNA.

Conclusion: Now that there is a slight band for secondary PCR, the next step in lab is to start PCR squared into order to amplify the DNA sequence.

Primary PCR (10-13-14 to 10-28-14)
 * Figure 1: (10-13-14) 1kb ladder is in well 1 and 10. My primary PCR is in well 8. There is a slight smear, but not compared to the other primary PCR of other students. **
 * Figure 2: (10-14-14) On well 6 is the 1kb ladder, on well 7 is Jairo’s primary PCR, on well 8 is my secondary PCR, but since there is not a band my experiment failed. **
 * Figure 3: (10-16-14) 1kb ladder and my primary PCR with a new oligo mix sample in well 1 and 3, respectively. **
 * Figure 4: (10-17-14) 1kb ladder, primary with 1ul the new oligo mix, primary with 2ul of the new oligo mix, and primary with 1ul of Q5 polymerase in well 2, 3, 4, and 5, respectively. **
 * Figure 7: (10-27-14) 1kb ladder, primary with 1ul jairo’s oligo mix, primary with 2ul of jairo’s oligo mix, and primary with 1ul of Q5 polymerase in well 2, 3, 4, and 5, respectively. [[image:dtn548_102814_AgaroseGelPrimaryPCRTargets_INVERT.JPG width="485" height="560"]] **
 * Figure 8: (10-28-14) 1kb ladder, primary with 1ul jairo’s oligo mix, primary with 2ul of jairo’s oligo mix, and primary with 1ul of Q5 polymerase in well 2, 3, 4, and 5, respectively. **

Analysis: In Figure 1, there was a slight band, so I tried secondary PCR, but as shown in figure 2 in well 8 there is not a band, so the experiment was a failure. Based on the fact that my experiments failure 4 times in a row, I made a new oligo mix just in case if I missed an oligo. In figure 3, the primary failed in well 3. I addressed this failure by making 3 samples: sample 1 with the regular amount of solutions for the primary PCR protocol, sample 2 with 2ul of the new oligo mix, and sample 3 with 1ul of Q5 polymerase. Once again, in Figure 4, there were no smears. Next, I used Jairo’s oligo mix using the same method I used in Figure 4, but as you can see in Figure 5 there were no smears. This failure could have been due to contamination in the agarose gel indicated in from the smug within the 1kb ladder. Therefore, I ran the same experiment from figure 4 and 5, but without contamination within the gel, and there were slight smears in Figure 6.

Conclusion: Now that there are clear smears in Figure 6, the next step in VDS is to conduct secondary PCR.

Week 5, 6, and 7 PCR (10-2-14 and 10-3-14)  Figure 1: Trial 1 of Primary PCR will my DNA in well 8.

 Figure 2: Trial 2 of Primary PCR will my DNA in well 4.

 Analysis: As shown in Figure 2 and 3, my primary PCR was a failure. In Trail 1, I used the Thermocycler cycling conditions in black, but the reason was probably that I put in the Q5 Polymerase in the sample far sooner than I should. In Trail 2, I used the Thermocycler cycling conditions in orange, but instead of the temperature of 58 Celsius I used a temperature of 62.6 Celsius, which is the melting point.

 Conclusion: As both the trail 1 and trail 2 primary PCR failed, the next step is to run another gel to see if it will work.

Midi-Prep (10-2-14)  Figure 1: Nanodrop trail 1 of pNIC-bsa4 yield



 Figure 2: Nanodrop trail 1 of pNIC-bsa4 yield

Analysis: If all goes well, the pNIC-bsa4 plasmid will be in the last 1.5 mL tube. Some of the DNA may have been stuck in the filter due to the mistake of drawing back the plunger with the filter still attached to the 5mL syringe.

Conclusion: The purpose of this lab is to take the pallet from transformation with pNIC-Bsa4 and clean it so that we will end up with only the plasmid. The average concentration of the finished product is 52.8 ng/uL. Once we have under go transformation and Midi-prep for the DNA of our target protein, the next step would be to insert the target DNA into the pNIC-bsa4 we purified today.

Primer Tail Design (9-29-14) <span style="font-family: Tahoma,Geneva,sans-serif;">DNA sequence of STP1 <span style="font-family: Tahoma,Geneva,sans-serif;"> 1 ATGGAGATCAGCCTGCTCACTGACATTGGCCAGCGTCGTTCTAACAATCAGGACTTCATC <span style="font-family: Tahoma,Geneva,sans-serif;"> 61 AATCAGTTCGAAAACAAAGCGGGTGTTCCGCTGATCATCCTGGCGGACGGCATGGGCGGT <span style="font-family: Tahoma,Geneva,sans-serif;"> 121 CACCGTGCGGGCAACATCGCTTCCGAAATGACCGTTACCGACCTGGGTTCCGACTGGGCG <span style="font-family: Tahoma,Geneva,sans-serif;"> 181 GAGACCGACTTCTCTGAACTGTCTGAAATTCGTGACTGGATGCTGGTATCTATCGAAACC <span style="font-family: Tahoma,Geneva,sans-serif;"> 241 GAAAACCGTAAAATCTACGAACTCGGTCAATCTGACGACTACAAAGGTATGGGTACCACC <span style="font-family: Tahoma,Geneva,sans-serif;"> 301 ATCGAAGCCGTAGCGATCGTTGGTGACAATATCATCTTCGCGCACGTCGGTGACTCTCGT <span style="font-family: Tahoma,Geneva,sans-serif;"> 361 ATCGGTATCGTTCGTCAAGGTGAATATCACCTGCTGACGAGCGACCACTCTCTGGTTAAC <span style="font-family: Tahoma,Geneva,sans-serif;"> 421 GAACTGGTCAAAGCAGGCCAGCTGACTGAAGAGGAAGCGGCGTCTCACCCGCAGAAAAAC <span style="font-family: Tahoma,Geneva,sans-serif;"> 481 ATCATCACCCAGTCTATCGGTCAGGCGAATCCGGTTGAACCGGACCTCGGTGTACATCTG <span style="font-family: Tahoma,Geneva,sans-serif;"> 541 CTGGAAGAAGGTGACTACCTGGTTGTTAACTCTGACGGTCTGACCAACATGCTGTCTAAC <span style="font-family: Tahoma,Geneva,sans-serif;"> 601 GCGGACATCGCGACCGTTCTGACCCAGGAAAAACCGCTGGACGACAAAAACCAGGACCTG <span style="font-family: Tahoma,Geneva,sans-serif;"> 661 ATCACCCTGGCGAACCATCGTGGTGGTCTGGACAACATCACCGTTGCGCTGGTTTACGTT <span style="font-family: Tahoma,Geneva,sans-serif;"> 721 GAATCTGAAGCGGTAAT <span style="font-family: Tahoma,Geneva,sans-serif;">Upstream: TACTTCCAATCCATGGAGATCAGCCTGCTCACTGA <span style="font-family: Tahoma,Geneva,sans-serif;">Downstream: CGTTGAATCTGAAGCGGTATAACAGTAAAGGTGGATA <span style="font-family: Tahoma,Geneva,sans-serif;">Reverse compliment: TATCCACCTTTACTGTTATACCGCTTCAGATTCAACG <span style="font-family: Tahoma,Geneva,sans-serif;">

Analysis: In order to get the original tail primers to get within 10 of each other I had to reduce the length of the forward tail primer from 35 to 29 nucleotides. One must have the melting temperatures of their tail primers to be closer to each other in order for them to function under the same circumstances. If the melting temperatures of the tail primers are too far apart then there is a possibility that one of the tail primers may denature since the other tail primer’s annealing temperature could be close to the melting temperature of the other.

Conclusion: The purpose of this lab is to design our own primers for pNIC-Bsa4 in order to amplify the production of a certain gene we are trying to get. Once we’ve designed and ordered the tail primers, it will be used in secondary PCR and in PCR squared.

<span style="font-family: Tahoma,Geneva,sans-serif;">PyMol Refresher (9-19-14) <span style="font-family: Tahoma,Geneva,sans-serif;"> <span style="font-family: Tahoma,Geneva,sans-serif;">Fig 1: Chain A of 2H2Q with ionic residues as blue, polar residues as light blue, hydrophobic residues as yellow, and NAP colored by element. <span style="font-family: Tahoma,Geneva,sans-serif;"> <span style="font-family: Tahoma,Geneva,sans-serif;">Fig 2: Chain B of 2H2Q with ionic residues as blue, polar residues as light blue, hydrophobic residues as yellow, and NAP colored by element.

<span style="font-family: Tahoma,Geneva,sans-serif;"> <span style="font-family: Tahoma,Geneva,sans-serif;">Fig 3: 3CL9 with ionic and polar residues as blue, hydrophobic residues as yellow, NAP as cyan, UMP as green, EDO as purple, MTX as red, and the active site as orange.

<span style="font-family: Tahoma,Geneva,sans-serif;"> <span style="font-family: Tahoma,Geneva,sans-serif;">Fig 4: 1U72 shown as green lines with its ligands NDP and MTX as red and yellow respectively. 3CL9 shown as cyan lines with its ligands NDP and MTX as blue and pink respectively.

<span style="font-family: Tahoma,Geneva,sans-serif;"> <span style="font-family: Tahoma,Geneva,sans-serif;">Fig 5: 3HBB with ionic and polar residues as blue, residues as yellow, NAP as red, TMP as green, EDO as purple, MTX as red, and the active site as orange.

<span style="font-family: Tahoma,Geneva,sans-serif;">Analysis: 1U72 AND 3CL9 are about 50% similar, but this could be due to the fact that 3CL9 has two chains while 1U72 has one. Otherwise, 1U72 and 3CL9 are very similar with there ligands bounded very similarly.

<span style="font-family: Tahoma,Geneva,sans-serif;">Conclusion: The purpose of this lab is to refresh the students on the procedures of PyMol in order to utilize it when we analyze our target protein. The next step when using pymol is to analyze docking from once virtual screening is finished and determine if ligands bound close enough to know inhibiters to our target protein.

=<span style="font-family: Tahoma,Geneva,sans-serif;">Week 3 and 4 =

<span style="font-family: Tahoma,Geneva,sans-serif;">PCR Figure 1: Agarose Gel of with 1kb lader in lane 2 and 7.

Analysis: As shown on Figure 1 the experiment was a failure. I pipetted at the lowest part off the well and the samples were pushed our, so the gel couldn't capture the DNA sequence.

Conclusion: The next step is to make more of the sample and run the gel again.

<span style="font-family: Tahoma,Geneva,sans-serif;">Restriction Enzyme Digest Figure 1: Agarose Gel of with 1kb lader in lane 2 and 7.


 * Analysis:** As shown on Figure 1, the experiment was a failure. This is most likely due to improper calculations; where the amount of ul of plasmid used is wrong. Instead calculations should have been **(1,500 ng/ul)/(972.9 np/ul) = 1.5417823ul.** Therefore, because there was such a small amount of the pGBR22 protein the EcoRI, PvuII, and EcoRI + PvuII samples were not shown in lanes 4-6.


 * Conclusion: ** The next step is to run the gel again with the right amount of pGBR22 to compare the EcoRI, PvuII, and EcoRI + PvuII samples.

Week 1&2 Analyzing DNA Sequence (9-5-15) <span style="font-family: Tahoma,Geneva,sans-serif;"> <span style="font-family: Tahoma,Geneva,sans-serif;">Figure 1: VDS4_M13R_pGBR22_060509forBB.txt WITH N’s <span style="font-family: Tahoma,Geneva,sans-serif;"> <span style="font-family: Tahoma,Geneva,sans-serif;">Figure 2: VDS4_M13R_pGBR22_060509forBB WITHOUT N’s

<span style="font-family: Tahoma,Geneva,sans-serif;"> <span style="font-family: Tahoma,Geneva,sans-serif;">Figure 3: First blast using the VDS4_M13R_pGBR22_060509forBB.txt WITHOUT N’s

<span style="font-family: Tahoma,Geneva,sans-serif;">

<span style="font-family: Tahoma,Geneva,sans-serif;">Figure 4: Second blast using the VDS4_M13R_pGBR22_060509forBB.txt WITHOUT N’s

<span style="font-family: Tahoma,Geneva,sans-serif;"> <span style="font-family: Tahoma,Geneva,sans-serif;">Figure 5: completed pGBR22 DNA sequence

<span style="font-family: Tahoma,Geneva,sans-serif;">Analysis:

<span style="font-family: Tahoma,Geneva,sans-serif;">Nanodrop (8-29-14) <span style="font-family: Tahoma,Geneva,sans-serif;"> <span style="font-family: Tahoma,Geneva,sans-serif;">Figure 1: Trail 1 of pGBR22 will a concentration of 257 ng/ul with a maximum absorbance of 5.139 at about 260 nm. <span style="font-family: Tahoma,Geneva,sans-serif;"> <span style="font-family: Tahoma,Geneva,sans-serif;">Figure 2: Trail 2 of pGBR22 will a concentration of 257 ng/ul with a maximum absorbance of 4.264 at about 260 nm.

<span style="font-family: Tahoma,Geneva,sans-serif;">Analysis: