MATERIAL FOR QUESTIONS 3-6 s Disease is caused by expanded CAG repeats in the HT
ID: 215962 • Letter: M
Question
MATERIAL FOR QUESTIONS 3-6 s Disease is caused by expanded CAG repeats in the HTT gene. You are studying HTT and decide to clone it downstream of Green Fluorescent Protein (GFP), so that you can tracik where HTT functions in the cell (e.g. at the plasma membrane, nucleus, mitochondria, etc.). McS 5500 bp Amp You have a plasmid that already has an enhancer and promoter upstream of GFP. The stop codon of GFP has been deleted and replaced with a multiple cloning sequence that has four restriction enzyme sites as shown in the plasmid diagram. The sites are engineered so that sequences you clone into the MCS will be "in-frame" with the coding sequence of GFP Ori As shown by the map below, HTT cDNA is 9435 bp long and contains restriction enzyme sites to EcoRI and Xhol. HTT CDNA does not have restriction enzyme sites for Spel or Sphl. TT DNA-9435 bp long Ir lacks restriction sites for Spel and Sph START ho EcoRl Ecol STOP 553 5030 9435 te: as you know, the DNA depicted in these diagrams is double-stranded I site GAGCT and the restriction enzymes that we are working with cut both strands. For example, as shown above and to the left, Xhol cuts HTT cDNA at position 508 downstream of the ATG start site at position 1. We refer to the cut agctegagctctataaggaaatt positions of the restriction enzymes based on the top strand only tcgagctegagatattcctttaa res 5..C'TCGAG..3 .ACTAGT. .3 5.GCATGc. Xhol 3 GAGCT pel 3.TGATCA.. 3.CGTACG EcoRI GAATTC 3.CTTAAG...5Explanation / Answer
A. Complete media with ampicillin. USing this media will allow for selection and growth of only transformed cells since plasmid has ampicillin resistance gene.
B. After 20 cycles number of bacterial cells= 2^20= 1048576
copies of plasmid= 1048576 X 10= 10485760 copies of plasmid since plasmid gets copied 10 times after each cell division.
C. There can two probable reasons for not getting any individual colonies but a lawn of bacteria
1. Either the transformation mixture after recovery which is used for spreading on the LB plate is too much. Since transformation efficiency is higher you should less transformation mixture or dillute it 100 times to get some individual colonies.
2. Other reason might be that you forgot to add Ampicillin in LB media so the colonies that you see on plate are both transformed and untransformed ones.
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