4 replies to this topic

[ariaz]

Hi,
I did PCR and got a 75 bp DNA  which was ok and then inserted it into topo vector (4 KB). I got colonies, i extracted DNA (Topo + insert) and digested it with Xho1 and Not 1 (because my insert had Xho1 site on one end and NOt 1 on other). I was not able to see any band of 75 base pair. I sequenced the Topo + insert and sequencing results were showing my insert correctly in Topo.
Please tell me the answers of these questions. I will be a great help.
I need to Know that how can i visualize and cut such small bp DNA from gel?
What percent of agarose gel will be good?
How much DNA should i use?
And if i want to gel purify this insert with normal Qiagan gel purify kits, will it be fine? because i heard that such small length DNA will not be attached to column.
Is there any way so i can purify the insert of this size?

ariaz

[Michaelro]

Hi
You may try 2% agarose gel in 1xTBE - I think it will be sufficient.
However, your prolem is most probably the amount of insert on gel.
Your insert is small - 75bp/4075bp = 0.018.
It constitutes only 1.8% of your plasmid.
Therefore, to obtain 100ng of the insert on gel, You should make a restriction on 100ng / 0.018 = 5500ng plasmid DNA.
I think if You cut a sufficient amount of the plasmid - You'll be able to see the insert.
Regarding the gel extraction kit, You should check the manual.
Usually, the manufacturer provides the information about the range of fragment sizes that will bind to the column.
For example with PureLink™ Quick Gel Extraction Kit (Invitrogen) You may purify fragments starting from 40bp.
Small fragments tend to bind with lower efficiency - ~50%.
Hope it helps
Good Luck
Michael

ariaz, on Jun 8 2010, 02:19 PM, said:

Hi,
I did PCR and got a 75 bp DNA  which was ok and then inserted it into topo vector (4 KB). I got colonies, i extracted DNA (Topo + insert) and digested it with Xho1 and Not 1 (because my insert had Xho1 site on one end and NOt 1 on other). I was not able to see any band of 75 base pair. I sequenced the Topo + insert and sequencing results were showing my insert correctly in Topo.
Please tell me the answers of these questions. I will be a great help.
I need to Know that how can i visualize and cut such small bp DNA from gel?
What percent of agarose gel will be good?
How much DNA should i use?
And if i want to gel purify this insert with normal Qiagan gel purify kits, will it be fine? because i heard that such small length DNA will not be attached to column.
Is there any way so i can purify the insert of this size?

ariaz

[ariaz]

Hi Micheal

Yes it will really help me. Thank you very much.
To get more of my insert i need more than 5 ug of plasmid. So i should probably maxi prep or midi prep my plasmid DNA so i can get this much of amount.
In Qiagen kit manual it is mentioned that fragment more than 70 bp will be eluted. I shall try to get the invitrogen kit which you mentioned.

Thanks again

ariaz

[HomeBrew]

If the presence, identity, fidelity, and orientation of your insert is confirmed by sequencing, why go to so much trouble to see it on a gel?

[ariaz]

Hi,
I need restriction digestion of my insert with the enzymes Xho1 and Not 1 and then insert it into another vector which has same enzyme sites in it. But each time when after digestion i run it on gel i couldnt find any band there. (may be due to smaller size of insert). Yesterday some one suggested me in lab that cut the gel exactly besides 75 bp band of  your ladder. (That is, run the sample and then under UV when you see ladder markers at 75 bp, cut the gel out of your sample exactly equal to it, whether there is a band or not). May be in this way i can get some thing.

HomeBrew, on Jun 9 2010, 04:04 PM, said:

If the presence, identity, fidelity, and orientation of your insert is confirmed by sequencing, why go to so much trouble to see it on a gel?