I'm trying to clone 3.0 Kb inserted into 5.5 Kb vector with one side that is blunt and one side that is a sticky end. After transformation, 20 times colony difference was observed between test and control (only vector) ligation. But when I tried to screen by PCR, no positives were observed. My PCR controls were fine. I checked by plasmid isolation also (5 colonies) and all were negative by digestion. I wonder what could be the reason behind this for not getting recombinants even though the colony difference was as expected?
Incomplete digestion of the vectors or carryover of the short fragment you cut. In this case, you might want to use CIAP to block sellf- or unexpected- ligation.
I am curious how your "negative" plasmids look like. Are they all look like an empty vector or are they smaller and in general have different sizes? Also did you get lower efficiency of transformation compare to your successfull clonings? Sometimes if you get a good difference between your ligation and negative control and the same time all "positive" clones are empty it can mean that your vector does not "stand" the insersion and only recombined molecules are surviving.
The colony cracking procedure is a technique for detection of inserted gene, and in comparison with colony PCR method is easier. The following is the recipe and protocol for this assay.
2X Lysis buffer for (colony cracking):
10 mL Sucrose (20% w/v), NaOH (1 M) 200 mM, KCl (1 M) 120 mM, EDTA 10 mM,
SDS (10%), 500 µL BPB (Bromophenol Blue)
*store at -20°C because the BPB degrades in alkaline condition
Protocol
Add 30 µL Lysis buffer in PCR tubes
Add one colony to each tube
Incubate at 37 °C for 5-7 min
Incubate on ice for 5 min
Centrifuge at 13-14 k for 10 min
Run 10-15 µL on agarose gel
Hope it helps.
It may b due to that ur ligation is not occuring properly. U have to first standarise ur ligation nd can confirm it after performing restriction digestion of the ligated product. U have to optimise the concentration of vector and insert
Use some otherethod like silver staining and page to view the insert in pcr. Might b conc is quite low to be viewed in simple agaroge gel
I used QIAGEN gel extraction kit.I tried colony PCR. No positives. I also fell that vector backbone ( from where I'm trying to take insert) is giving this kind of profile. This I could trace by plasmid preparation of some of the colonies. With this kind of result, Insert ligation will definitely give more colonies. I tried to remove the vector back bone contamination by another gel extraction. Now even in gel I'm confident that my insert is not contaminated with the vector backbone. I repeated the transformation with the fresh preparation of vector and insert. This time another problem , control ligation( only vector) gave more colonies than the test ligation ( vector+insert). I'm trying to trace out this. I request your opinions on this issue.
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