Find the genetics map in part A and B and answer the question. What were the ste

Question

Find the genetics map in part A and B and answer the question. What were the steps or information placed in the Plasmapper in the “User Defined Features” for each section A and B to get the genetic maps? Just confused how to get the maps with the program.

PlasMapper

http://wishart.biology.ualberta.ca/PlasMapper/

PlasMapper

For our analysis we will use an online application, PlasMapper, that generates and annotates circular plasmid maps. Taking only the plasmid/vector DNA sequence as input, PlasMapper recognizes common replication origins, antibiotic resistances, restriction sites and other features and produces a graphical or textual description.

PlasMapper

http://wishart.biology.ualberta.ca/PlasMapper/

Part A

Go to this site and paste the DNA sequence for the empty pCR2.1 plasmid (sequence 1) and click on graphic map. This is a map of your pCR2.1 plasmid without any insert. Go back to the form and add a “User Defined Features” that will display the location of the M13 (-20) Forward priming site (DNA base pair numbers for this “feature” can be found in the screenshot). Create a new graphic map with this feature. Paste as answer.

Part B Now we will add our PCR product to the vector and make a new map. To do so you need to know where the PCR product will be inserted into the vector. You can find this information by looking at the plasmid map (pCR2.1_Map) The pCR2.1 vector is not a closed circle until an insert is ligated into it. Each of its DNA strands has an overhanging T (thymine) on the 3’ end. The PCR amplification of the insert puts an extra (overhanging) A (adenine) on the 3’ end of each of the insert DNA strands. This allows the extra A’s on the 3’ ends of the PCR product to match up with the overhanging T’s of the pCR2.1 vector. The overhanging T’s of the pCR2.1 vector are located between two Eco RI sites. To help you, here is part of the plasmid sequence that shows the two EcoR1 sites in bold. Note that we are not cutting at these Eco RI sites in the pCR2.1 plasmid – they are just helpful in finding the site where the insert will ligate in.

GAATTCGGCTT___PCR INSERT___AAGCCGAATTC

The sequence of the whole plasmid with the EcoRI sites highlighted can be found below. You must copy the PCR sequence from what you entered into webcutter above and paste it into the correct site in the plasmid. Now create a new map with the PCR product as part of the plasmid. Hint…you need to figure out the base pair numbers for the PCR product once inserted into pCR2.1 and create a new “User Defined Feature” for the PCR product. Past picture as answer. Question: What happens to the nucleotide numbering of the shown plasmid features when the PCR product is inserted? If you wanted to keep your user defined feature of the M13(-20) forward priming site, how would you change the numbering to make sure the labeled site was accurate?

pCR2.1 (3929 bp). Showing Eco RI sites. Sequence starts at base pair defined as #1 AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTT TCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAG GCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAA ACAGCTATGACCATGATTACGCCAAGCTTGGTACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGC TGGAATTCGGCTTAAGCCGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAG GGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGA AAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAA GAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGG CGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCC GCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGG GGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGG TTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAAT AGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGA TTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAA AATTCAGGGCGCAAGGGCTGCTAAAGGAAGCGGAACACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGAC CCCGGATGAATGTCAGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAGC TTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCA GCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAGGA TCTGATGGCGCAGGGGATCAAGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGA TGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACA ATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCG ACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGT TCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCG GGGCAGGATCTCCTGTCATCCCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGC GGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACG TACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCC GAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCT GCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGC GGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGAC CGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACG AGTTCTTCTGAATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTT GCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGT TGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGA AGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCC GGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAG AAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACAC TGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGG GATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACA CCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTC CCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCG GCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGG GGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACG AAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCA TATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATA ATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCA GCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGC AGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCC TACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGG TTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGC CCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAG CTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGAT TTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCT GGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATT ACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGG AAGCGGAAG

acZa ATCG Hind III M13 Rewerse Primer CAG GAA ACA CT ATGA C ATG ATT AC CCA AGC TTG GTA cia A Toa GAT COA CTA G TAC TAA T GGT TOG AAC CAT TCG AGC CTA GGT GAT GTA ACG GOC GCC AGT GTG CTG TTC GGC TT Ava Xhol PCR Product T CGG CTT AAG ACG PaeA? BstX I NsI Xba AGA TAT CCA TCA CAC Taa con oca CTC GAG CAT GCA . TCT AGA AAT TOG COC TAT Dra Il site M13 Forwerd (20) Primer AGT GAG TOG TAT TACAAT TCA CTG GOC GTC GTT TTA CAA CGT OGT GAC TGG GAA AAC TT GCA GCA CTG ACC CTT TTG GTTA AGT pCR 2.1 3.9 kb Commenta for pGR 2.1 3929 nucleotides gene: baaes 1-545 : baaes 205-221 M13 Reverse priming aite T7 promoter: bases 362-381 M13 (-20) Forward f1 origin: bases 546-983 Kanamycin resistance ORF: bases 1317-2111 Ampicillin resistance ORF: bases 2129-2989 pUC origin: baaes 3134-3807 priming site: bases 389-404

Explanation / Answer

Part A

In order to generate a pCR2.1 plasmid map with highlights of the feature M13(-20) forward primer, you just identify its sequnce location in total plasmid sequence. The start and end point of the primer sequeces are 389 - 404 (given in screenshot). Therefore, to solve part A, paste the plasmid sequence of pCR2.1 and then go to "User defined features" , enter Feature 1 name as " M13(-20) forward primer", enter category as "tag", and then enter start as "389", stop as "404", and strand as "Reverse". Then click "Graphic map" option to generate pCR2.1 plasmid with required feature.

Part B

You know the basepairs or sequence length of your PCR product. For example if your PCR product length is 300bp with overhangs, you can directly ligate the product in to EcoR1 sites. Your PCR insert is flanked two sides with EcoR1 restriction sites. Now you number the features that follows next to the First EcoR1 site by adding the insert size.

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