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A59BB1 Molecular Biology for Bioinformatics I Continuous Assessment Task 1: Answer Sheet

A detailed restriction map of pBR322, which was needed to answer questions 2
and 3, is provided at the end of this document. This particular map has been
taken from the catalogue of a company (New England Biolabs) that supplies
materials for molecular and cell biology.
1. The fragments of genomic DNA have been inserted into the BamHI site of
pBR322. Since the BamHI site lies within the tetracycline resistance gene of pBR322, insertion of DNA into the BamHI site will inactivate the tetracycline resistance gene. Consequently, ampicil in would have to be used to select for transformants. [3 marks]
2. pBR322 is 4.4 kbp in size. From the results of the restriction enzyme digests, we can conclude that the recombinant plasmid is 10.1 kbp in size, so the inserted DNA must be 10.1 - 4.4 = 5.7 kbp in size. We can map the vector EcoRI and PstI sites in the recombinant plasmid from the information given in the restriction map of pBR322. This is shown in Figure 1 (a). Although the DNA in the recombinant plasmid has been inserted into the BamHI site of pBR322, we cannot be certain at this stage that there is a BamHI site at either end of the insert. This is because Sau3AI fragments were used to digest the genomic DNA from the pathogenic bacterium. Ligating a Sau3AI site to a BamHI site may produce a BamHI site, but not necessarily. EcoRI digest first. There are two fragments, which tells us that there is one EcoRI site in the insert. This site must lie 2.9 kbp from the EcoRI site on the left of the restriction map, as shown in Figure 1 (b). The EcoRI site in the insert cannot be 7.2 kbp from the EcoRI site on the left of the restriction map, because then it would be located outside of the insert. Now consider the PstI digest. As with the EcoRI digest, there are two fragments, indicating again that there is one site within the insert. This site must be located 7.1 kbp to the left of the vector PstI site, as shown in Figure 1 (c). If it was 3.0 kbp to the left of the PstI site, it would still be located in the vector. BamHI sites. Referring to the BamHI and EcoRI “double” digest, note that there is a 6.1 kbp fragment. The only possible explanation for this is that a BamHI site lies 6.1 kbp from the left of the EcoRI site on the right of the restriction map, as shown in Figure 1 (d). This predicts a 1.1 kbp BamHI-EcoRI fragment, which was obtained in the restriction digest. Now consider the 2.5 kbp fragment. It must be located 2.5 kbp to the left of the EcoRI site in the insert, as shown in Figure 1 (e). This predicts a 3.6 kbp BamHI A59BB1 Molecular Biology for Bioinformatics I fragment, which was obtained in the digest with BamHI alone. In other words, a BamHI site has almost certainly been produced by ligation of the vector BamHI site to the Sau3AI site of the insert. This predicts a small BamHI-EcoRI fragment of 0.4 kbp, and indeed this was found in the double digest. The final restriction map is shown in Figure 1 (f). [12 marks]
3. The restriction map predicts sizes of 1.2, 1.8, 1.8 and 5.3 kbp. Always check your working by making sure that the fragments add up to the expected size of the plasmid (10.1 kbp in this case). On an agarose gel, this digest would actually appear to consist of only three fragments, because there are two fragments each of 1.8 kbp. However, the fact that the sum of the fragments would only appear to add up to 8.3 kbp, combined with the greater intensity of the 1.8 kbp fragment, would (or should) alert the investigator. [5 marks]
4. Sau3AI is a “four-cutter”, whereas BamHI is a “six-cutter”. Consequently, there will normally be more Sau3AI sites than BamHI sites in any given sequence of DNA. Therefore, a partial digest with Sau3AI would produce a more random digest of the genomic DNA, compared to a partial digest with BamHI. This will provide a higher proportion of overlapping fragments of DNA, and a greater probability of cloning a complete sequence of interest. [5 marks]
A59BB1 Molecular Biology for Bioinformatics I
Figure 1. Restriction map of the recombinant plasmid. The box indicates the
insert DNA. Dashed lines represent the vector/insert boundaries. B, BamHI; E,
EcoRI; P, PstI. Numbers are kbp.
pBR322 is an E. coli plasmid cloning vector containing the Open reading frame (ORF) coordinates are in the form origin of replication from pMB1 (a plasmid in the ColE1 "translational start – translational stop”; numbers refer to See page 132 for ordering information.
compatibility group; 1–3). The rop gene product, which positions on the top (clockwise) strand, regardless of the regulates plasmid replication by stabilizing the interaction direction of transcription and include the start and stop codons.
between RNAI and RNAII transcripts, maintains the copy Origin of replication coordinates include the region from the -35 number at about 20 per cell. However, pBR322 can be promoter sequence of the RNAII transcript to the RNA/DNA amplified with chloramphenicol or spectinomycin (4).
switch point. bla (ApR) gene coordinates include the signal Enzymes with unique restriction sites are shown in bold type
and enzymes with two restriction sites are shown in regular Coordinates
type. The accompanying table shows restriction sites of those enzymes that cut a moderate number of times. Restriction site coordinates refer to the position of the 5´-most base on the top strand in each recognition sequence.
ori = origin of replicationAp = ampicillin, Tc = tetracycline EcoR I - Apo I 4359
Aat II 4284
Cla I - BspD I 23
Hind III 29
EcoR V 185
Ssp I 4168
BamH I 375
SgrA I 409
Sca I 3844
Pvu I 3733
EcoN I 622
Pst I 3607
Sal I - Acc I - Hinc II 651
PshA I 712
Ase I 3537
Bsa I 3433
Ahd I 3361
BspM I - BfuA I 1063
PflM I 1315
Bsm I 1353
AlwN I 2884
Ava I - BsoB I 1425
Msc I 1444
rop
Bsg I 1650
Pci I - Afl III 2473
BspE I 1664
BsaB I 1668
Sap I 2350
Pvu II 2064
Nde I 2295
BsmB I 2122
BstZ17 I - Acc I 2244
BsaA I 2225
Tth111 I - PflF I 2217
References
1. Bolivar, F. et al. (1977) Gene 2, 95–113.
2. Sutcliffe, J.G. (1979) Cold Spring Harb. Symp. Quant. Biol. 43, 77–90.
3. Watson, N. (1988) Gene 70, 399–403.
4. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Source: http://web.sls.hw.ac.uk/teaching/bioinformatics/_2005/mbfb01/files/A59BB1_Task1_Answer.pdf

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