Category Archives: Results

Preparation of successfully amplified ycf1 from DNAs 4-34 to 4-44

On March 23, 2012 Tyieshia Rhodes and Canda’Ce Elleby used exonuclease and alkaline phosphAtase enzymes to remove primers and dNTPs from DNA samples 4-34 to 4-44. After the primers and dNTPs were removed, we used the Nano-Drop technology to amplify all of the DNA samples. Below Table 1 shows the sufficiently high yields that were obtained.

Table 1: PCR products yields

PCR products ng/ul
4-34Y 303.16
4-35Y 318.85
4-36Y 398.32
4-37Y 470.61
4-38Y 347.9
4-39Y 336.69
4-40Y 428.87
4-41Y 390.31
4-42Y 383.13
4-43Y 458.06
4-44Y 364.15

Preparation of successfully amplified xdh from DNAs 4-1 to 4-11

After analyzing the size of our DNA bands that were amplified on the gel, we moved forward with 10 of our 11 DNA samples, excluding 4-10. Our PCR results, (refer to https://armstronggenetics3.wordpress.com/wp-content/uploads/2012/03/rr-tk-xdh1.jpg) show high concentration yield with bright bands for samples 4-1, 4-2, 4-4, 4-5, 4-6, 4-8, 4-9 and moderate to low yield with broad bands for samples 4-3, 4-7, 4-11.  All PCR products only had bands specific for the amplified region in our nuclear gene xdh.  DNA purification of our 10 PCR products was accomplished by, using Exonuclease I to destroy the remnant primers,Fast Alkaline Phosphatase to inactivate the dNTPs, and incubation at 37*C.  The DNA purification results were quantified on 03/23/2012. These values are listed below  in Table 1.

Table 1: DNA Quantification via NanoDrop Spectrophotometer

Sample ID ng/ul
4-1X 463.4
4-2X 392.81
4-3X 414.57
4-4X 435.84
4-5X 526.93
4-6X 528.74
4-7X 530.3
4-8X 535.09
4-9X 512.13

Preparation of Successfully Amplified ycf1 from 4-23 Through 4-33

On March 23, 2012, Brittany Edge and Courtnee Pettus used Exonuclease and alkaline phosphatase to clean up and remove primer and  dNTP’s from the samples that were successfully amplified. We were able to clean up 10 out of the 11 DNA samples from 4-23 through 4-33. However, sample number 4-27 was not successfully amplified because it did not yield a high enough amount. Therefore it was not able to be included in the purification and quantification processes. A visual of this PCR gel can be viewed by referring to the following link,https://armstronggenetics3.wordpress.com/2012/04/06/ycfi-from-4-23-through-4-33/. Here you will find a picture of the gel and as you can see in lane number six, sample 4-27 did not produce a high enough yield. After the successful yields were treated with Exonuclease and phosphatase, they were incubated at 37­°C and then by using the Nano-Drop Spectrophotometer, the samples were quantified. Below in table 1 you will find the results of the purification process which includes the sample numbers and their yields after purification.

Table 1: Nano-Drop Spectrophotometer Yield Results

Sample ID       ng/ul
4-23Y 428.54
4-24Y 476.58
4-25Y 407.27
4-27Y 479.11
4-28Y 455.87
4-29Y 436.02
4-30Y 452.58
4-31Y 374.87
4-32Y 391.44
4-33Y 418.03



Clean-up of Successfully Amplified petB From DNA 4-1 through 4-9

Brandon Jones and Duong V. performed a PCR cleanup, seven out of the nine DNA’s were cleaned successfully. Two of the samples were poorly amplified (4-5 and 4-8) and were not able to be used in the PCR cleanup results as illustrated in Table 1. The PCR results in Table 1 shows moderate to high yield for samples: 4-1, 4-3  and a high yield for samples: 4-2, 4-4, 4-6, 4-7 and, 4-8.  The cleanup process was performed by using Exonuclease I and Alkaline Phosphatase combined with PCR mixture for further purification.Our results shown in Table 1 below are from using the nano-drop spectrophotometer.

Table 1: Quantification of the enzyme treated PCR products

Sample ID

ng/ul
4-1B

389.01
4-2B

496.81
4-3B

366.41
4-4B

463.28
4-6B

557.95
4-7B

521.77
4-9B

545.84


Clean-up of Successfully Amplified petB from DNA Codes 4-19 Through 4-27

Upon completion of DNA gel analysis of our PCR products, we found that 5 out of the 9 samples were shown to have high or moderate yield. The samples of high yield were 4-23, 4-24, 4-25, and 4-27. The sample of moderate yield was 4-21. Samples 4-20, 4-22, and 4-26 failed to give any yield. With the use of Exonuclease I and Alkaline Phosphatase, we “cleaned” the products of sufficiently high yields of any excess material other than the petB intron. Table 1 below contains the results of DNA purification.

Table 1: DNA Quantification by NanoDrop Spectrophotometer

Sample                                                Yield (ng/ul)

4-21B 376.27
4-23B 412.25
4-24B 414.8
4-25B 391.32
4-27B 385.13


Preparation of successfully amplified petB from DNAs 4-28 to 4-36

After analyzing our PCR products via a DNA gel, we moved forward with 8 of the 9 samples, excluding 4-30 (Diaphananthe pellucida) as we did not detect visible product for this sample (no band was visible on the DNA gel). Our PCR results show high yield for samples: 4-28, 4-29, 4-34, 4-35 and moderate to low yield for samples: 4-31, 4-32, 4-33, 4-36. All products only had bands specific for the petB intron. By using Exonuclease I and Alkaline Phosphotase with incubation at 37°C, we purified our 8 samples of excess primers (single stranded DNA) and dNTPs. Our quantified DNA purification results are illustrated in the following table (Table 1).

Table 1: DNA Quantification via NanoDrop Spectrophotometer

Sample ID ng/ul Date
4-28B 388.19 3/22/2012
4-29B 373.6 3/22/2012
4-31B 404.16 3/22/2012
4-32B 417.56 3/22/2012
4-33B 381.19 3/22/2012
4-34B 369.74 3/22/2012
4-35B 376.22 3/22/2012
4-36B 402.79 3/22/2012

Preparation of successfully amplified atpF from DNAs 4-1 through 4-11

Steven Jackson and Jenavieve Rummel performed a PCR “clean-up” of atpF from DNAs 4-1 through 4-11. By referring to previous PCR results, atpF from DNAs 4-5 and 4-10 were omitted from clean-up due to the low yield. The remaining products 4-1 through 4-11 were treated with Exonuclease I and “FastAP” alkaline phosphatase. Nano-Drop Spectrophotometer quantified the results. The results are shown in Table 1: atpF.

Table 1:atpF
Sample ID                             ng/ul                            Date
4-1F

466.30

3/22/2012
4-2F

533.50

3/22/2012
4-3F

507.90

3/22/2012
4-4F

456.47

3/22/2012
4-6F

474.65

3/22/2012
4-7F

488.70

3/22/2012
4-8F

489.14

3/22/2012
4-9F

424.19

3/22/2012
4-11F

370.66

3/22/2012

Preperation of successfully amplified Ycf1 from DNAs 4-1 to 4-11

Table 1: Yield (ng/µL) of products (ycf1 4-1 to 4-11) were collected by using nano-drop technology (2nd column). Date of collecting data was recorded (3rd column).

Mary C., Carly S. and Tram N. performed a PCR cleanup experiment using Nano-drop technology on ten of the original eleven DNA samples, excluding 4-10 (lane 11, refer to PCR post https://armstronggenetics3.wordpress.com/category/results/pcr-data/ycf1/). According to table 1. we obtained a high yield of products, previously evident in our gel (thick bands denote high product concentrations) for the PCR that followed initial DNA extractions. It was necessary to exclude DNA sample 4-10 because of its failure to produce an adequate amount of product, evident by the lack of band in the gel. This may be due to error on our part since, when Carly S. ran the gel she neglected to submerge it in buffer. However it is most likely that the DNA extraction was ineffective and the sample was inadequate to the point that no amplification occurred.

This Post was a collaborative effort of Mary C. and Tram N.

Preparation of successfully amplified atpF from DNA’s 4-12 through 4-22

Sherri Field Gard and Melissa Fry were assigned the targeted region atpF in the samples 4-12 to 4-22.  In the previous gel electrophoresis of the given samples, it was determined that samples 4-14 and 4-20 produced insufficient yield. These samples were excluded from further inclusion in the research project.  The remaining samples from 4-12 to 4-22 showed strong single bands of correct size and were cleaned up using the Exonuclease-Alkaline Phosphatase kit and provided protocol.  Samples were then placed in the Nano-drop for quantification of product.  High yields were noted, as shown by the results in Table 1.  Samples were then prepared for shipment by Dr. Jarrell, and exported for DNA sequencing.

Table 1
Sample ID ng/ul Date
4-12F 351.85 3/22/2012
4-13F 353.47 3/22/2012
4-15F 362.52 3/22/2012
4-16F 363.79 3/22/2012
4-17F 377.02 3/22/2012
4-18F 318.02 3/22/2012
4-19F 360.58 3/22/2012
4-21F 351.52 3/22/2012
4-22F 344.25 3/22/2012