4 replies to this topic

[kariskalon]

Hey there,
super noob undergrad student here.
Let alone ligation, I am having hard time preparing my vector.
I successfully cut and purified my insert (1.2kbp) using gel extraction and etOH ppt but when i did the same with my vector, i basically got 7ng/uL in NanoDrop. The following is what I tried.

1) First, digested ~5uL of vector with 1ul EcoRI HF and dephosphorylated with 1uL dephosphatase at the same time for ~15 min.
2) heat inactivated at 65C for 20 min.
3) double digested with 1uL of 1/10x XhoI (BSA added) and 1uL of 1/10x ndei for an hour (EcoRI was just to prevent religation).
4) heat inactivated at 65C for 20 min.
5) ran on 1% gel at 37V for an hour -- the band was clean, bright, and the right size.
6) giagen gel extracted (under 30 sec UV exposure)
7) etOH ppt
- added 1/10 volume of DNA sample of 3M sodium acetate and 2.5x vol of 100% etOH
- stored at -80C for 2 hours
- spun at 15000g for 40 min
- decanted most of supernatant, dried at 60C till no liquid was visible
- saw a clear pellet and resuspended in miniprep elution buffer and heated for 10 min at 60C.
NanoDrop= 7ng/uL!! ((
i followed almost the exact steps for my insert and it worked..

Also, i tried double digesting with XhoI (BSA) and NdeI while dephosphorylating all at the same time, heat inactivated for 20 min at 65C then used Cyclepure microelute to clean up the enzyme and small fragments, etc. then i got yet again another ~7ng/uL. This method failed me twice. is it ok to use PCR clean up kit for vector after digestion? this was in attempt to avoid gel extraction.

Could it be that the clear pellet was merely salt and i actually got rid of my DNA?
then how would i explain the second situation with the kit?
i ended up using my labmate's vector with my insert for ligation. still have to do transformation...
i've been having problems with vector digestion and i'm afraid ligation would be far far away.
any advice would be appreciated and thank you for reading!

[cellthetruth]

First, what is the amount of vector you digest initially? I suggest to use around 3 ug in total. Second, you should be aware that the gel-solving buffer is not that nice to your DNA, so incubate just that long until your gelfragment is dissolved!

Then, try this precipitation protocol:
- Add 0.1 vol 3M sodium acetate and 3 vol 100% EtOH
- store at -20°C over night
- spin max. speed, 20 min
- remove supernatant liquid with pipette, avoid touching the pellet
- wash with 70% EtOH
- spin max. speed, 20 min
- remove supernatant liquid with pipette
- AIRDRY your pellet, do not overdry as the pellet gets very hard to resuspend!
- resuspend your DNA in 20 ul dH2O or Tris-HCl pH 7.5

for me this works fine. I get something around 100ng/ul, i.e. approx. 2/3 of initial DNA.

[phage434]

7 ng/ul of DNA is perfectly fine to do a ligation and transformation.  You don't need much, and quality is far more important than quantity.  Dilute your insert so that you can have equimolar amounts of insert and vector, ligate, transform and go.

[kariskalon]

thank you both for reading my long post and responding
i did my digestion again today and got low yield after EtOH ppt (had enough after gel extraction) once again.
i guess it's due to lack of my lab technique (it's hard working with clear pellet)
but i will have to work on that and also try using the low yield that i have.

thank you!

[bob1]

Gel extractions always give a low yield.  As Phage said - you don't need much for a ligation - 20 ng of vector is heaps.

You also don't need your initial EcoR1 digestion - Nde1 and Xho1 are incompatible ends, and also shouldn't need dephosphorylation.  If the fragment you are cutting out is less than about 50 bp, then you don't need a gel extraction - PCR extraction kit or EtOHpptn will be fine as the small fragment will not be bound by the column.  You may find it best to do the digestions sequentially rather than simultaneously, and to test the REs individually to make sure that they are cutting well.