L. Briefly describe how we wil be able to select for bacterial colonies that con
ID: 198113 • Letter: L
Question
L. Briefly describe how we wil be able to select for bacterial colonies that contain pC vectors with lambda DNA inserted into the polycloning site 2. Why do we need to include the control plates mentioned in part Ill of the protocol? What wil it mean if bacteria grow on these plates? What will it meani no bacteria grow on these plates Read through the protocol for this week's lab and determine how many plates you will need. List what you will be plating on each one Pretecol: Each team will perform the following protocol three times using the fellowing plasmids 3) uncut puC Plpet 40 ul DH5a competent cells into a clean mcfg tube. 2. Add 1 DNA (recombinant plasmid or uneut puC. 3.Gentily mix by pipetting up and down a Sew times Pipet the miture into an electroporation cuvette Tap gently to make sure there are no bubbles and thur the lquid is at the bottom of the chamber. Dry the outside of the cuvette 4 Place the cuvette into the electroporator 5. Sen the electroporator to 1800 V. Press "pulse two times 6 After the beep, check the bime constant-it should be around 4-5 7 Remove the cuvette and immedately poet 600 . LB media intothe cuvette. 8 Pipete the 640 ul into a stenle tube. 9 incubate with shaking at 37 for about 45 minutes. 0 Wash out the cuvettes that you used by flushing once with ethanol twice with D flask and treat with bleach Invert the washed cavettes on paper towe to dry .Preparing the plates Our TA has prepared plates containing LB agar and 200m/ampidlinfor selection ofthe pUCiS vector. We need to add X-gal and IPTG to make blue/white screening possible. I wil demonstrate the technique for spreadingX-gal and IPTG on the plates 1 Label the necessary number of platesclearly so that you can identity them later, and aso label to indicate that you wil have added X-gal and IPTG 2. Evenly spread 40 of X-gal + IPTG onto each plate 3. Allow the liquid to soak in for 15 minutes before going on IL·Plating the electroporated cells I will demonserate this technique. l will plate some untransformed DHSa cells ona plain LB a plate and an LB agar plate+ amp. Why are these control plates necessary? You will want to plate each of your samples of transformed cells on two separate plates-one plate with 30of cells and second plate wth 100 . of cels. This is toensure that you will be able to identity and iselate separate colonies. 1. Label your plates so that you know which ceills you are plating and what volume of those cells you are plating. 2. Plpet the cells into the center of the plate and spread evenly 3. Allow the liquid to soak in for 15 minunes. 4 Invert and incubate at 17'C overnight S. The next day come in and check your plate. Record your observations in your lab notebook using th, table below as guide. Aho record observations for our two control plates with untransformed DHSa cells. Place your plates on the counter and I will store them in the fridge until next week Volume plated |Kind of plate -ofwhite LB agar LB agar+ ampExplanation / Answer
1- colonies in which pUC vector containing lambda DNA will be white in color whereas colonies which have only pUC vector will appear blue in color.
2- In order to check whether the untransformed cells doesn't have any ampicillin resistance. As these colony will also show white color which can be misinterpreted as false positive. If no bacteria grows on the plate then we can confirm that the untransformed cells don't have any amp resistance.
3-
We will need following plates-
Plane LB Agar plate - Untransformed cells
Agar plate + Amp - untransformed cells
Agar plate + X-gal- untransformed cells
Agar plate + Amp+ X-gal - untransformed cells
Agar plate + Amp+ X-gal - transformed cells
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