heres some background) A cloning experiment was being perfomred where the C. ele
ID: 207719 • Letter: H
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heres some background)
A cloning experiment was being perfomred where the C. elegans unc-54 gene had undergone PCR, than restriction enzyme digest, after which it was ligated into a plasmid vector Puc19. after ligation it was transformed into a group of E.coli where two screening techniques were undergone, ampicillin resistance and a blue white screening., aafter leaving the plates for 24 hours to cure the next day all that was seen was blue and white colonies of E. coli, A gel miniprep was undergone where 3 white colony and 1 blue colony had there DNA purified and ran on a gel electrophoresis, and that is what is seen here, im not sure about what is significant about or how to interpret this? going from left to right wells are Ladder, white colony, white colony white colony, blue colony, and Ladder
here are the steps to prepare the minipreps incase anyone wondered, they didnt involve a restriction enzyme digest, from what i can expect,
1 Pellet 1.5ml of your overnight culture in a 1.5ml microfuge tube, spin 13,000rpm for 1 minute. Remove the supernatant by pipetting it off.
2 Resuspend the pellet completely with 250l of Resuspension Solution(R3): pipette up and down and vortex the tube.It is important that it is completely resuspended.
3 Add 250l of Lysis Solution (L7) to each sample:mix gently by invertingthe tube several times until it looks clear and viscous.
4 Add 350l of PrecipitationSolution(N4); shake hardabout 4 or 5 times to mix, a white precipitate should form and the solution seem less viscous.DO NOT VORTEX
5 Centrifuge at maximumspeed(~13,000rpm)for 5 minutes at room temperature, most of the precipitate will be pelleted, there may be a little floating on the surface.
6. Set up a Spin Column into a Collection Tubeand label the column, not the tube.
7. Pipette the cleared lysate carefully into the Spin Column, avoid sucking up any of the precipitate.
8. Centrifuge at top speed for 1 minute at room temperature. Discardtheflow-through, and reinsert the Column into the Collection Tube.
9 Add 500l of Optional Wash Buffer(W10, ethanol added). Incubate at room temp for 1 min. Centrifuge at top speed for 1minute. Discard the flow-through
10. Add 700l of Wash Buffer(W9, ethanol added). Centrifuge at top speed for 1minute. Discard the flow-through.
11.Centrifuge the empty column at top speed for 1minute to remove all traces of the Wash solution.
12.Transfer Spin Column to a sterile 1.5ml microfuge tube.
13.Add 75l of TE to the middle of the Spin Column. Incubate 1 minute at room temp then centrifuge at top speed for 2minutes.This is your eluted DNA.
14.Discard the column, label the tube with what it is and your name.
Take 5ml of each of the purified DNA samples and run them on the agarose gel 5ml water and 3ml 6xloading dye first). Don’t forget to include the 1Kb ladder. Run at 100v 30minutes. Image
The gel under UV light with the Gel Imager. Note the sizes of the bands, which is larger and why
THANKS ALOT
6000 3000 1000Explanation / Answer
My suggestion to this promblem will be : what i see here in the band double in size for the ladder DNA on right side as compared to ladder DNA on left side. The right side ladder DNA has not separated or resolved properly as compared to the left side ladder can be the possible explanation.The probable reason for this could be the amount/quantity of ladder DNA the study have used.The quantity of ladder DNA used optimal is around 10 l for 1KB. The question have not fished out or mentioned the concentration the quantity. So my suggestion will for using this concentration.Attaching refernces for these which can be helpful.Refernce: Susan Carson, D. Scott Witherow, in Molecular Biology Techniques (Third Edition), 2012.
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