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 intron from 4-37 through4-44

Table 1
PetB
Sample ID	ng/ul	Date

4-37B	502.92	3/22/2012
4-38B	465.8	3/22/2012
4-41B	448.43	3/22/2012
4-42B	551.9	3/22/2012
4-44B	460.72	3/22/2012

Jennifer Walden and Blake Jones performed a PCR clean up on the five (4-37-4-44) successful yields from PCR, as seen in the chart above.  Using Exonuclease I and Alkaline Phosphatase combined with PCR mixture, we performed a clean up reaction for additional purification.  All five strong bands yield high numbers using a Nano-Drop Spectrophotometer acquired the data above.  The three samples not used for clean up were very weak or not visible, with little product, which may have been due to human error.