Hello everybody,
Does anyone have an experience with direct colony sequencing?
Last month, I transformed TOPO® Cloning reaction into TOP10 cells. I analyzed positive transformants using Colony PCR and I also extracted their plasmids using Qiaprep Miniprep but when I measured their concentration I found that all of them were around 10 ng per μL. I need at least 100 ng per μL for sanger sequencing. I have extracted their plasmids several times using different protocols but the results were not satisfying. So, do you think direct colony sequencing is suitable for me?
(After sequencing I want to transform my plasmids into BL21.)
Here is other information:
The size of my insert is : 550 bp
Name and size of my vector: pET151/D-TOPO® 5760 bp
Strain: TOP10
Primers for sequencing : T7 and T7 Revers
Thank you
if you can pcr amplify across the entire insert from a colony then you have plenty of pcr product to sequence ( pcr product is shorter than the plasmid so you need less product) and the only problem should be that you cannot sequence the first 30 bases after the primer so you may have to move the primers out from the insert or sequence in both directions to get the entire sequence
if you can pcr amplify across the entire insert from a colony then you have plenty of pcr product to sequence ( pcr product is shorter than the plasmid so you need less product) and the only problem should be that you cannot sequence the first 30 bases after the primer so you may have to move the primers out from the insert or sequence in both directions to get the entire sequence
Many thanks Paul for your reply,
I am considering using T7 and T7 reverse primers for sequencing and their priming sites are out from the inserts ( around 100 bp).
But my concern is that whether direct colony sequencing is appropriate or not because my plasmid is low copy number.
Hi, if you need these plasmids for further experiments why not to increase volume of cells (2-3 times) used for plasmid propagation. You can apply lysates to same purification column, decrease elution volume and easily get yield ~50ng per uL, 50 or 100 is not so crucial for Sanger sequencing.
I do not think that copy number matters much as you double the amount of pcr product every cycle so you can just run more cycles or use nested pcr starting with primers outside of T7 then re amplify with the T7 primers
Sara Heidarpanah apart from what said before, if you still not get desirable results, yes, you can sequence colony directly. There are several companies which can provide the service. Google such company which might suit you you their prescribed protocol.
I would agree with Paul Rutland that rather than do direct colony sequencing, why not just amplify the region you wish to sequence and directly sequence that PCR product. Much easier and more reliable to do.
I have tried to use PCR from direct colony PCR but results were very bad with high background.
At these time I used both F and R primer for each fragment to cover both ends.
If I were you, I will use 10 to 20 ml culture and concentrate cells before mini prep. to get more DNA concentration of my plasmid then sequence it.
Another choice is to use PCR would could anneal 100 up and 100 down from your target sequence, then use same primers for sequencing your PCR fragment.
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