Hi all,
I've been struggling with transformation procedure for past few months. First of all, I'm working with producing chimeric genes using Overlap Extension PCR. Parallel to this, I have been working with 2 single genes also. After the chimeric gene(around 1.5kb) is PCR amplified, it is again subjected to Agarose Gel Electrophoresis and then purified using QIAquick gel extraction kit. The restriction enzyme digestion image is attached here. The wells include: 1) NEB 1kb DNA ladder 2) Single gene double digest with around 1.5kb 3) and 4) Chimeric construct double digest with 1.5kb size 5) Another single gene double digest around 2kb size 6) and 7) are double digests of pUC19(1000ng) 8) Single digest of pUC19(250ng) using BamH1 9) Single digest of pUC19(250ng) using EcoR1. For double digest, BamH1 and EcoR1 are used. Restriction digested DNA was heat inactivated at 65 degree for 20minutes. After gel extraction, the ligation was kept with two systems: A) 5ng vector with (1:3,1:5,1:7 insert) B)10ng vector with (1:3,1:5,1:7 insert).
After the transformation, no colonies were visible even after blue white screening. Only in plates of single gene with 2kb showed at least blue color, even though it's negative result. Also, the growth in plates of competent cells showed lawn growth and uncut vector showed ample amount of growth. In our lab, subcloning is advised, like first cloned in pUC19(DH5a) to pET28a BL21(DE3) cells
Can anyone help me with suitable suggestions? I've been trying for months and I'm desperate to get a clone. Kindly suggest some troubleshooting steps.
Is it really just 5 ng and 10 ng of vector? That's probably the reason why you aren't getting colonies. Not enough DNA.
Typical cloning guidelines are 50 ng of vector for every 3 KB of vector size (so if you have a 6 KB vector that's 100 ng of vector into your ligation)
Amy Klocko Thank you for your reply! Actually we are using DH5α competent cells. And other scholars in my lab, got colonies in the same ratio for different genes but with kind of same length and also, their clone has been confirmed by sequencing. So, I'm confused where I'm going wrong. Last time when I repeated the experiment, I kept controls for ligation like: 1) Double digested vector with ligase 2) Double digested vector without ligase. But surprisingly, both didn't give any colonies. But when I re-checked where I went wrong, I added double digested insert DNA also in the ligation control(1). Basically, there was no control (1). I'm going to do transformation with your suggestion also. Can you please clarify if the total DNA content should be 100ng or just vector content should be 100ng? Thank you! :)
Hello Arsha,
You can get colonies with less DNA, but if the ligation and/or transformation efficiency gets too low then you risk getting no colonies.
To clarify, the 100 ng is for the vector backbone. It's 100 ng vector plus however much insert is 3X moles of the vector.
Here are the reactions I run when I'm cloning something new
digestion controls of the vector
uncut
single cut
single cut
double cut
You can't really see a cut difference on inserts that are also PCR products, but might be worth a check to make sure you aren't getting unwanted cutting
ligation controls
double digested pure vector with no ligase and no insert (checks for how much was uncut to begin with)
double digested pure vector adding the ligase but no insert (checks to see if you got self-ligations of single cut vector)
then the actual ligation, double digested pure vector plus ligase plus insert
50 ng vector per every 3 kb vector size, 3x as many moles of the insert
Oh, as a note, BamHI can have star activity, even in the correct buffer. It can be kind of a pain to clone with. If you have NEB BamHI, it used to cut extra sites in buffer 3 on some vectors (I had issues with pCAMBIA)
good luck!
Amy Klocko For the restriction digestion we are using BamH1-HF from NEB. I've used pET28a for double digestion and added Shrimp Alkaline phosphatase(rSAP) from NEB along in the restriction digestion reaction. In NEB protocol, they mentioned, we can add along with restriction digestion . So I followed that also. Is there any other method for this? for adding alkaline phosphatase?
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