Simply Cloning - Chapter 9 - Clone Verification

To verify that our plasmids have the insert that we wanted to introduce, we are going to cut them with XhoI and BamHI, and separate the resulting DNA fragments on an agarose gel.
Clone verification protocol:
1. Restriction mix
o 16.5 µl ddH2O
o 1 µl miniprep DNA
o 2 µl 10X restriction buffer
o 0.25 µl restriction enzyme A (2 units)
o 0.25 µl restriction enzyme B (2 units)
2. Incubate at 37 oC for 45 min
3. Run on an agarose gel, take a picture
Right now I am going to set up a restriction digest with the plasmids that we just isolated. For one reaction I will need:
• 16.5 µl ddH2O
• 1 ul of plasmid DNA
• 2 µl of NEB Buffer 3
• 0.25 µl of XhoI
• 0.25 µl of BamHI
I also prepared three new Eppendorf tubes for the restriction reactions.
In order to avoid extra pipetting, I am going to prepare a master mix, and then split it into three aliquots, one for each of the plasmids.
In the master mix I am going to put three times the amount of all reagents:
• 49.5 µl of water
• 6 µl of NEB Buffer 3
• 0.75 µl of XhoI
• 0.75 µl of BamHI
I will mix up the master mix and put 19 microliters of it into each of the new tubes. Then I will add 1 µl of plasmid to each of the reaction tubes, and mix them up by pipetting.
As usually, I am going to incubate restriction digests at 37 oC for 45 min. Then I will run the cut plasmids on an agarose gel.
Let's have a look at the gel picture.
You can see that for each of the clones I run uncut vector along with the cut one. In the case of clone two, where there is no fragment coming out, uncut lane allows me to see if any digestion took place.
In the case of clone one, we have three bands: one for linearized vector, one for vector without insert, and one for the insert itself. Also this gel allows us to verify the size of the linearized vector and of the insert.
The bottom line here is that both clones 1 and 3 contain inserts and the inserts are of the correct size.
Hey, we got the clone!
One last thing we have to do, is to put this precious clone in a special corner of refrigerator known as the Clone Library.

[@balajishinde9743]

wonderful

[@alexduval4740]

I did the calculations for Clone 1 for "Cut" vector lane and it is log(3845)= 3.584(without Bar insert), log(4395)=3.642(with Bar insert) and log(550)=2.74(the Bar fragment). This is very consistent on the log graph with the observed distances these respective linear DNA's migrated which is around 320 mm (without Bar insert), 340(with Bar insert) and 615 mm (the Bar fragment). Good work.