Using the plasmid maps for orientation 1 and 2 that you filled out, find the exp
ID: 213398 • Letter: U
Question
Using the plasmid maps for orientation 1 and 2 that you filled out, find the expected sizes for all of the possible single digestion schemes and fill out the table below (Hint: If you cut a plasmid in orientation 1 with BclI, you get 3 fragments—one is 302bp—you can figure out the size of the other two. Because the starting plasmid is 5541, fragments will always sum up to this starting value).
Orientation 1:
Orientation 2:
Fill out this chart:
cDNA FRAGMENT (1622bp) A 3 3' A 5' (1)(17) (319 (665) (720(750 1025) (1622) This is the cDNA fragment that has been ligated into the TOPO site of the pSRP plasmid. It is 1622bp and contains 3'A overhangs. The numbers represent the location of each restriction site in relation to the left side of the fragment. The A overhangs are NOT included in the length of the cDNA The cDNA FRAGMENT can be ligated into the pRSP plasmid in two distinct orientations Flip 1800 (735) (1642) (1651) (1662 (1662) (680) (6) (32) (334) o40 pRSP + cDNA-5541bp 2615 Bcll (orientation 1) Kanamycin Res. Note that the ends of the cDNA fragment have NO polarity meaning that the 3 and 5' ends are exactly the same The same is true for the ends of the pRSP after it is cut with TOPO (see figure 42). Thus, either end of the cDNA can be ligated to either end of the pRSP Since many copies of the TOPO cut plasmid are mixed with many copies of the cDNA insert during the ligation reaction, there will be TWO ways that the cDNA can be ligated into the pRSP The figure above shows ONE of the orientations and the location of each restriction site. Note that the size of the new pRSP plasmid is now 5541bp and the restriction sites within the multiple cloning site that were to the RIGHT of the ligation site are now 1622bp away from the EcoRi site that was to the left of the ligation siteExplanation / Answer
cDNA orientaion
1
2
1
2
1
2
1
2
1
2
BClI
BClI
ECOR1
ECOR1
ECORV
ECORV
Xbal
Xbal
XhoI
XhoI
No of sites
3
3
3
3
2
2
3
3
1
1
Expected no of fragments
3
3
3
3
2
2
3
3
1
1
Size of fragment 1(bp)
302
302
759
887
916
725
360
360
5541
5541
Size of fragment 2(bp)
2281
995
877
755
4625
4816
632
700
Size of fragment 3(bp)
2958
4244
3905
3899
4549
4481
Sum of fragments (bp)
5541
5541
5541
5541
5541
5541
5541
5541
5541
5541
cDNA orientaion
1
2
1
2
1
2
1
2
1
2
BClI
BClI
ECOR1
ECOR1
ECORV
ECORV
Xbal
Xbal
XhoI
XhoI
No of sites
3
3
3
3
2
2
3
3
1
1
Expected no of fragments
3
3
3
3
2
2
3
3
1
1
Size of fragment 1(bp)
302
302
759
887
916
725
360
360
5541
5541
Size of fragment 2(bp)
2281
995
877
755
4625
4816
632
700
Size of fragment 3(bp)
2958
4244
3905
3899
4549
4481
Sum of fragments (bp)
5541
5541
5541
5541
5541
5541
5541
5541
5541
5541
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