Archive image from page 68 of The development of a cosmid. The development of a cosmid map of chromosome 12p13 . developmentofcos00belk Year: 1998 half the signal of K562 DNA because it contained only one copy of the chromosome where K562 DNA contains two copies. The positive band for cl70G6, a cosmid that was previously mapped to the same STS marker, allowed for verification of the methods being used. These being verified, further testing could be undertaken. Determining the Placement of Marker D12S1914. To determine the relationship between markers D12S950 and D12S1914, a PCR react
Archive image from page 68 of The development of a cosmid. The development of a cosmid map of chromosome 12p13 . developmentofcos00belk Year: 1998 half the signal of K562 DNA because it contained only one copy of the chromosome where K562 DNA contains two copies. The positive band for cl70G6, a cosmid that was previously mapped to the same STS marker, allowed for verification of the methods being used. These being verified, further testing could be undertaken. Determining the Placement of Marker D12S1914. To determine the relationship between markers D12S950 and D12S1914, a PCR reaction was run using cl70G6 with primers 950F, 950R and cl28D6 with primers 1914F, 1914R. A positive band was observed for cl28D6 at approximately 75 base pairs but no band was observed for cl70G6 (FIGURE 17). Figure 17. PCR of cl70G6 with forward and reverse 950 primers and of cl28D6 with forward and reverse 1914 Primers. Lanes 2 and 8 contain 1KB ladder ( \ig). Lane 3 contains cl 70G6, Lane 4 is a negative control of TE for 950 primers. Lane 5 contains an unknown cosmid suspected of being cl70G6, Lane 6 has cl28D6, and Lane 7 contains a TE negative control for 1914 primers. In Lane 6 there is a visible positive band for cl28D6 with marker D12S1914 at approximately 75 base pairs. PCR with Cosmid Pools. In order to process the cosmid DNA more efficiently, cosmid pools of 5 L each of five different cosmids were prepared for 94 of the available cosmids for the section of chromosome 12 being mapped. In general testing with cosmid pools was unsuccessful. Positive controls did not show bands at migration distances corresponding to the STS markers being tested. Agarose gels did not run smoothly or photograph clearly, giving fiizzy banding overall or light primer bands and no marker bands at higher molecular 33
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