Bio 181 Restriction Mapping Tutorial

Transcription

1 Bio 181 Restriction Mapping Tutorial You have cloned an unknown cdna 3 kb long into the EcoRI site of vector plasmid pbluescript (also 3 kb). You need to prepare a restriction map of the cdna insert to set up your future experiments. This is what you know about the ligated plasmid before you start: MCS = multicloning site Unknown cdna insert 3 kb Left half of MCS: EcoRI PstI SmaI BamHI XbaI NotI SacI amp R pbluescript (vector) 3 kb ori Right half of MCS: EcoRI EcoRV HindIII ClaI SalI XhoI ApaI KpnI The insert is cloned into the Eco RI site of the multicloning site (MCS/polylinker). You have this restriction map of pbluescript, so you know what enzyme sites are on the extreme right and left of the insert. As with all polylinker restriction sites, these sites are unique in the vector. You first cut the plasmid with all of these polylinker enzymes to see which of them cut in the insert, and where the sites are relative to the site in the vector. You set up restriction digestion for each of the enzymes and allow them to cut for one hour. After the incubation period, you load them on a gel. You run the digestions from the left side on one gel and the right side on another gel. 1

2 Left Side Enzymes Marker EcoRI PstI SmaI BamHI XbaI NotI SacI Uncut

3 Right Side Enzymes Marker EcoRI EcoRV HindIII ClaI SalI XhoI ApaI KpnI Uncut

4 To interpret the results, first determine whether each enzyme has a cut site within the insert (you already know each enzyme cuts within the vector s MCS). Based on whether the enzyme as zero, one, or more than one site in the unknown cdna insert, list the enzymes names in the appropriate category below. Next, list the sizes of the bands that they produce. No Sites Single cutters Multiple Sites Sma I Pst I: 2.1 kb BamHI: 4.2 Xba I 3.9 kb 1.2 Not I 0.6 Sac I: 3.1 EcoRV 2.9 Cla I Sal I Apa I HindIII: 4.3 KpnI: All produce 1.0 a single 6.0 kb Xho I: 4.6 band 1.4 Bands must add up to 6.0 kb (size of whole plasmid) Make estimates of smallest bands first; gel size precision is poor at the higher levels. Add up to 6.0 See maps at end of this document. HINTS: When estimating the sizes of the bands, note that the sizes of fragments larger than the 2.3 kb marker are hard to estimate and therefore inaccurate. Rely more on the sizes of the smaller bands and make sure that the sum of all the bands equals the total plasmid size (6.0 Kb). If you see only one band and it's 6.0 Kb (3.0 Kb of insert and 3.0 Kb of vector) then put that enzyme under "No Sites." If you see two bands, put that enzyme under "Single Cutters". List the fragment sizes. If you see more than two bands, put that enzyme under "Multiple Sites". List the fragment sizes. 4

5 Mapping Single Cutters: The enzymes that fall in the first category, No Sites in the cdna are already mapped - they don't exist in your insert. The digestion cut the plasmid only once, in the polylinker, and it was linearized. The enzymes that fall in the second category have one site inside your insert; two bands resulted from one cut in the polylinker and one inside the insert of the circular plasmid. The size of the smaller band is the distance from that enzyme's site in the polylinker to its site in the insert. The larger band is the part of the insert left on the vector after the digestion plus the vector plasmid (3.0 Kb). For example, if enzyme X cuts in the right MCS and gives a 2.0 Kb band and a 4.0 Kb band, then the site for X in the insert is 2.0 Kb from the right side. If enzyme Y cuts on the left side and gives a 0.5 Kb band and a 5.5 Kb band, then the insert site for Y is 0.5 Kb from the left side: Figure 2 On the back of this page, draw a restriction map of the single cutters. Label each enzyme s cut site in the vector s MCS, and the location you deduced for the cut site in the unknown cdna insert. 5

6 Mapping Multiple Cutters: The appearance of three bands in a lane on the gels on pages 2 & 3 means that the enzyme cuts in three places. One cut is in the polylinker, which is part of the vector plasmid, so its position is known - either on the left side of the insert or the right. To map the two additional cut sites inside the insert is difficult. The location of one site can be identified from the gel data you have: The vector plasmid is 3.0 Kb. The largest fragment is larger than 3.0 Kb. Since there are no other sites for this enzyme in the vector, the largest fragment must contain the vector plus some of the insert. Therefore, the second site is the size of the large fragment minus 3.0 kilobases from the opposite side of the polylinker. That is, if the multiple cutter is on the left and you have a 4.3 Kb fragment, then the second site is 1.3 kb from the right side. At this point, there are two possible positions for the final cut site that could account for the two other DNA fragments seen on the gel. For each of the multiple cutter enzymes, draw two maps. Each map should definitively locate one of the cut sites within the insert, and should show one of the two possible locations for the second cut site within the insert. At present you do not have enough data to correctly identify the second cut site within the insert. In order to locate it, the plasmid must be double cut: with the enzyme being mapped, and with an enzyme that has a single, known cut site in the insert. To pin down the location of the second cut site, choose an appropriate second enzyme. Do a double digest, and look for disappearance of a band (cleavage into two smaller ones) compared with the single digest. {Details in class} 6

7 1300 HindIII 1600 XhoI 600 KpnI kb insert 1800 BamHI 2100 Pst I 0/6000 EcoRI PstI SmaI BamHI XbaI NotI SacI Whole plasmid 6.0 kb 3.0 kb vector 2900 Sac I 3000 EcoRI EcoRV HindIII ClaI SalI XhoI ApaI KpnI 4500 Unspecified: 2 nd BamHI site ; 2 nd KpnI site. First, determine the two possible locations. For BamHI, these are at 600 & 1200 For KpnI, these are at 1600 &

8 To determine which second site is correct: Perform a double digest, using a second enzyme that will give you information about the region in question (in this case, I chose HindIII for both; other choices are possible). Run single digest of Bam or Kpn side-by-side on a gel with double digest and look for disappearance of a band. Only pay attention to the two smaller bands which are created by the mystery cut site; the large band, while it may be cleaved, is irrelevant for this analysis. BamHI BamHI + HindIII BamHI + HindIII 4.2 kb 1.2 kb 0.6 kb (if Bam site is at 600) (if Bam site is at 1200) KpnI KpnI + HindIII KpnI + HindIII 3.6 kb 1.4 kb 1.0 kb 0.7 kb 0.3 kb (if Kpn site is at 1600) (if Kpn site is at 2000) 8