Question

Name: Cloning Worksheet chromosome qut 756bp pBiochem 5.983 bp ladder Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 BamHI (4233) (230) IT Enzyme recognition S ..GATATC. 51 EcoRV S .. . 3..CTATAC... 5 12 Pvull 89 BamHI Pett You are given the task to clone yfg into the plasmid pBioChem. Follow the steps below. 1. The plasmid map for pBioChem is shown above. As a control, you run it out on a DNA agarose gel prior to enzymatic digestion. Draw the uncut plasmid in Lane 1. 2. In order to verify you have the correct plasmid you perform a double digestion of pBioChem with BamHI and Pvull. • How many fragments do you get? • What are their sizes? 14.11 bp. 45726 Draw in on the agarose gel on Lane 2.

very lost on this whole worksheet and i dont know if my current answers are even right, please help i need this done by today

Answer 1

Ary - The length of plamid to the band appear 5500 and 6500 hp ladder lined 1.2 13 is 5983 bp in between lade der band. 14 15 16 7000 6soo 5500 3 Co 2000 ISOO uso Ary Any figure I - we get 3 pragments - The sizes are 1421 1920 and 17sobp. for band see the gel Lone 2. ② - you get 2 fragments The sizes are loo be and 5875 bp. EcoRv gets Blunt end Pots gives sticky end y The pagments are as follows.

ECORV - pstt wa -GAT -CTA ATC- TAG - CTGCA -GACET- СтсH ATC- TAG - for cleavage fragment se gel image figt 2 5 I puth enzyme is most efficient becaus it provids shirky end, → Ligation process is also eary in sticley ends. Any 9 The PCR product as digestion with EcoRv and pots -СТ, САМ SATGE TAG per is product → The of digested length see the fight for lame 4, bando in

- Any s Puth T56pb sople Elara BAMHI 31 Bb construct. puull Bom, The size of newly cloned planned is 6631 bp. for determinig whether insest es present are not we can do double digestion with EWRV and est to IF our gene is cloned weget two baghent one is 756 bp and other one is 58 75 Bp. self ligation gf There is no chance of becaun one end is blunt other end is sticky. So if we digest impty we get the band one and other is 5875 bp. planmid is 108 bp