Yashar Behnam Andy Kim Jarry Xiao Ian Zhou

Three Day Gel Lab

Intro to Restriction Enzymes:

Which ones of these enzymes would leave blunt ends? The enzymes that would leave blunt ends are AluI and SmaI. Which ones would leave sticky ends? The enzymes that would leave sticky ends are EcoRI, HindIII, BamHI, and HhaI. (EcoRI) Are they sticky or blunt? The new DNA ends produced by EcoRI are sticky ends. (SmaI) Are there any hydrogen bonds between the cut sites? There are no hydrogen bonds between the cut sites because the cut produces blunt ends. Only sticky ends cut hydrogen bonds between cut sites. (SmaI) Are the new ends sticky or blunt? The new DNA ends produced by SmaI are blunt ends. (HindIII) Are these ends sticky or blunt? The new DNA ends produced by HindIII are sticky ends. HindIII (Strand 3, back): 5 AGCT 3 EcoRI (Strand 4, front): 3 TTAA 5 Are the base sequences of the HindIII and EcoRI tails complementary? No, the base sequences of these two tails are not complementary. This is because the two DNA

strands were cut by different restriction enzymes. EcoRI (Strand 1, back): 5 AATT 3 EcoRI (Strand 4, front): 3 TTAA 5 Are they complementary? Yes, the base sequences of these two tails are complementary. This is because the two DNA strands were cut by the same restriction enzyme. Imagine that you cut a completely unknown DNA fragment with EcoRI. Do you think that the single-stranded tails of these fragments would be complementary to the singlestranded tails of the fragments from Strip 1 and Strip 4. The single-stranded tails of these fragments would be complementary to the single-stranded tails of the fragments from Strip 1 and Strip 4 because the same restriction enzyme (EcoRI) is used to cut both DNA strands. Because EcoRI only cuts at the nucleotide sequence of 5 GAATTC 3, the two fragments must be complementary. Do you think it would be easier for DNA ligase to reconnect two fragments cut by EcoRI or one fragment cut by EcoRI with one cut by HindIII? What is your reason? It would be easier for DNA ligase to reconnect two fragments cut by EcoRI because the base pairs are complementary. By having complementary base pairs, the two strands will be more easily joined, and will not have any bulges (two purines) or dents (two pyrimidines). What would be the product of a digestion with the two enzymes EcoRI and EagI? The products of a digestion between EcoRI and EagI would create a linear fragment of 942 bases and another fragment containing 4,599 bases. What would be the product of a digestion with the two enzymes HindIII and ApaI? The products of a digestion between HindIII and ApaI would create two linear fragments containing 2004 and 3537 bases. What would be the products of a digestion with the three enzymes Hrndlll, Apal,and PvuI? The products of a digestion between Hrndlll, Apal,and PvuI would create three linear fragments containing 2004, 2880, and 657 bases. If you took the digestion products from question #1 and digested them with PvulI, what would the products be? The products of a digestion with PvulI would create two linear fragments of 2305 and 2294 bases. The 942 linear base fragment would not be digested because there is no code for PvulI to splice the DNA in this fragment.

Restriction Mapping of Plasmid DNA:

1. Estimate the sizes of the DNA fragments (in base pairs) by comparing them with the lambda/PstI size marker. These estimated sizes do not have to be exact. Sizing of the smaller fragments will be more accurate than sizing of the larger fragment. HpaI: 4000 + 550 = 4550 HpaI/PstI: 2000 + 1400 + 550 = 3950 HpaI/SspI: 2500 + 550 + 200 = 3250 HpaI/PstI/SspI: 1900 + 1450 + 550 + 200 = 4100 2. Determine the total size of the digested DNA by adding up the sizes of the fragments from each digest. You may take an average size from the four digests. The same DNA was digested in each sample, so the fragment sizes from the different digests should add up to the same total. (4550 + 3950 + 3250 + 4100)/4 = 3962.5 3. There are two HpaI sites present. Based on the number of fragments obtained from the HpaI digest, is this DNA linear or circular? Draw the DNA with the HpaI sites present. Based on the diagram, we see that HpaI obtains 2 fragments. Because HpaI has only 2 sites, the DNA must be circular. If the DNA were linear, there would be 3 different fragments with 2 sites. On circular DNA, two cuts only forms 2 pieces.

4. How many PstI sites are present? Because we know that HpaI has two sites, PstI must only have one site. This is evident in DNA sample HpaI/PstI which only has three fragments. We know that in circular DNA each site produces one fragment. If two sites are already taken up by HpaI, only one site is left for PstI to form the third fragment. 5. Where is the PstI site? Draw the position of the PstI site on the plasmid, relative to the HpaI sites. The PstI site is located at approximately 1950 nucleotides

6. How many SspI sites are present? Because we know that HpaI has two sites, SspI must only have one site. This is evident in DNA sample HpaI/SspI which only has three fragments. We know that in circular DNA each site produces one fragment. If two sites are already taken up by HpaI, only one site is left for SspI to form the third fragment. 7. Where is the SspI site? Draw the position of the SspI site on the plasmid, relative to the HpaI sites. It might be best if this is done in a separate sketch from the PstI sketch since we have not yet determined where the SspI and the PstI sites are relative to one another. The SspI site is located at approximately 750 base pairs.

8. Will the 600-bp HpaI fragment remain unchanged after digestion with either Pstl or Sspl? (Check the gel.) Based on the position of the fragments on the gel, the 600 base pair fragment of HpaI is unaffected by the two other enzymes. 9. Which fragments are unchanged from the HpaI/Pstl digest to the Hpal/Pstl/Sspl digest? Which fragments disappeared? Why did those fragments disappear From the HpaI/Pstl digest to Hpal/Pstl/Sspl digest, the 2000, 1400, and 550 base pair fragments remained unchanged. None of the fragments disappeared but one was added at 200 base pairs. 10. Which fragments are unchanged from the HpalI/SspI digest to the HpaI/PstI/SspI digest? Which fragments disappeared? Why did those fragments disappear? From HpalI/SspI digest to the HpaI/PstI/SspI digest, the 550 and 200 base pair fragments remained unchanged. The 2500 base pair fragment disappeared because the addition of PstI caused the fragment to be shortened to about 1900. 11. Is there a fragment that appears only in the HpaI/PstI/SspI digest? What does this

mean? There is no fragment that appears only in the HpaI/PstI/SspI digest. This means that the enzymes of HpaI, PstI, and SspI spliced the DNA in their respective locations/codes. Because there are no other fragments present, HpaI, PstI, and SspI were the only restriction enzymes used to digest this enzyme. 12. Draw the full plasmid map, with all restriction enzyme recognition sites present in their relative locations.

Practice Plasmid Mapping:

1. Examine your stained gel on alight box. Well 1: pMAP/PstI/SspI Well 2: pMAP/PstI/Hpal Well 3: pMAP/PstI Well 4: Lambda/PstI Well 5: pMAP/PstI/Hpal/SspI

2. Assign sizes to the lambda DNA/Pstl size marker bands on your gel. These marker bands are 514, 805, 1093, 1159, 1700, 1986, 2140, 2450, 2838, 4700, 11497 bp in size. Remember small DNA fragments migrate more quickly than large ones. 3. Now assign approximate sizes to the DNA fragments of unknown size by comparing them to the lambda DNA/PstI size marker. These approximations will not be perfectly accurate. That is all right since the exact sizing is not required for determination of the number and relative positions of the cut sites of the restriction enzymes.

4. Determine the total size of the digested DNA by adding up the sizes of the fragments from each digest. You should take an average size from the four digest: pMAP/PstI, pMAP/PstI/HpaI, pMAP/PstI/SspI, and pMAP/PstI/HpaI/SspI. Remember the same DNA was digested in each sample so the fragment sizes should always add up to the same total. ((1995 + 1582 + 602) + (2261 + 597) + (3239 + 580) + (1979 + 824 + 548)) / 4 = 3551.75 Number of PstI sites: Number of SspI sites: Number of HpaI sites: