You have to grow 5 ml of Ampicillin resistant E.coli. Ampicillin is required at
ID: 81805 • Letter: Y
Question
You have to grow 5 ml of Ampicillin resistant E.coli. Ampicillin is required at 50 mu g/ml in LB Ampicillin stock solution concentration will be _________________. How much Amp stock to add to 5 ml LB? ________________ 5 ml bacteria will be grown in a 25 ml conical flask. Yes/NO Circle one. 5 ml bacteria can be grown in a _______________ For this, where does the inoculum come from? _________________ 4. 2 X PBS contains 20 mM sodium phosphate, 300 mM NaCl per this study What is PBS abbrev mean? ________________ 10 x PBS is provided to you. Make 300 ml of 2 x PBS solution. The Lysis buffer is 20 mM sodium phosphate, 300 mM NaCl and 0.5 mg/ml lysozyme. The stock of Lysozyme given to you is 50 mg/ml Make 25 ml of Lysis buffer using PBS stocks from Q4.Explanation / Answer
A stock solution of 50 mg/ml will be reasonable. Because in that case, you need to add 5 microliter stock solution to be added which is 0.1% of the total volume. Therefore, volume change can be neglected. For a lower concentration of stock solutions, higher volume needed to be added which will introduce significant change in total volume leading to change in antibiotic concentration.
Using a too high concentration of stock solution such as 100mg/ml will require 2.5 microlitres to be added in 5 ml culture which is too low and might incorporate error.
Calculation: Final concentration 50 microgram/ml
total amount of antibiotic to be added in 5 ml culture= 50 X 5= 250 microgram
50 mg/ ml=50 microgram/ microliter
Therefore, 5 microliters of antibiotic from the stock to be added in 5 ml of culture.(5X 50= 250 microgram)
Yes, 5 ml culture can be grown in 25 ml conical as the ratio of the volume of culture and volume of flask should be ideally 1:5
5 ml of bacteria can be grown in the Test tube also. In that case, a starter culture is required to inoculate. Inoculum can not be directly taken from a colony of the plate. Grow the colony from the plate in LB broth and inoculate in final medium.
PBS: Phosphate Buffer Saline
How to make 2x 300ml PBS from 10x stock.
V1.S1=V2.S2
V1X10x=300 X 2x
V1= 60 ml
Therefore, 60 ml PBS from 10X stock can be prepared by taking 60 ml from 10X stock and adding 240 ml water to it.
Total amount of lysozyme to be added in 25 ml 2X PBS= 0.5X25= 12.5 mg
Stock solution is 50 mg/ ml. Again,
V1.S1=V2.S2 here S1= 50mg/ml and S2=0.5 mg/ml and V2=25 ml
V1X50=0.5 X 25
V1= 0.25 ml or 250 microlitre
250 microlitre is an insignificant volume to 250 ml. So. 250 microlitre of lysozyme stock will be added to 25 ml 2X PBS.
If you want more accuracy, you can add 250 microlitres of lysozyme stock to 24.75 ml of 2X PBS.
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