Problems with BbSI treatment - px459 vector

[Poornima Manavalan]

Hi all,

I have two questions:

a. I am using the px459 vector, and I used 30 minutes, 1 hr and 2 hrs incubations at 37C for BbSI treatment. When I run my gel (0.8%), there are two bands for cut vector, very close to one another and shows some smearing. The uncut vector looks fine on the gel. The cut seems to have two bands.

Can someone suggest what could be going wrong? Has anyone had this problem?

b.I did a gel purification of the cut px459 vector and used that for ligation. Also since this vector is big, how long should the ligation reaction be? I am using the T4 quick DNA ligase, and ligation is at RT for 15-20 minutes. After transformation, I dont see any colonies or my plate or my transformation efficiency is too low.

Should the T4 quick ligation buffer be stored in aliquots since it contains ATP?

Thanks

Poornima

[Alex RD]

HI, Bblues

what kind of transform method you using? heatshock?.. you can try electroporation, which will gave much higher positive result.

another option, you can prolong the ligation time for 16C overnight

good luck

在 2014年7月1日星期二UTC-4下午2时31分55秒，Bblues写道：

[Alexandre Rodrigues]

And yes, you should aliquot the buffer

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[A Duckworth]

Can you perform the one step digestion-ligation reaction with this plasmid?

http://www.genome-engineering.org/crispr/wp-content/uploads/2014/05/CRISPR-Reagent-Description-Rev20140509.pdf

I got loads of colonies with the pX330 using this protocol...For my experiments I just used the ligation buffer instead of the fastdigest+ATP+DTT.

Andy

[A Duckworth]

Also, might help if you have a plasmid with ampicillin resistance that you know works as a positive control for transformation...

Andy

On Tuesday, 1 July 2014 19:31:55 UTC+1, Bblues wrote:

[Bblues]

Thank you Andy and Alex. I am trying the 2 step digestion and ligation today which I found on the forum, keeping fingers crossed!! 

If you did combine digestion and ligation reactions, how long do you incubate at 37C?

 The other thing I am trying regular ligase instead of quick ligase, maybe something funky happening with PEG in quick ligase.

Poorni

[hopkins405]

Hi, i tried the combined digestion ligation and it worked really well (18/18 by sequencing!)
this is the protocol I used:

1. Oligo annealing without phosphorylation:

oligo 1 (100 microM)  2 ul
oligo 2 (100 microM)  2 ul
10x ligase buffer         2 ul
H2O                           14 ul
for a total volume of 20 ul, heat block at 95 degrees for 5 minutes, then turn off heat block with tubes inside to slowly decrease temperature

2. Digestion/ligation reaction:

PX459                                                                1  ul (100 ng)
annealed oligos from above                               1 ul (0.5 microM)
BbsI Fast Digest Thermo Scientific (FD1014)    1 ul
Fast Digest buffer 10X                                        2 ul
T4 ligase ( NEB, MO202S)                                 1 ul
H2O                                                                  14 ul
total                                                                   20 ul

37 degree waterbath for 2 hr, heat shock 2 ul into bacteria--lots of colonies the next day,  all correct!

thank you all for your posts!

[Bblues]

Thats awesome!! It worked for me too, just tried it! I am sticking to this protocol, it saves so much time and no gel purification :)

[JORGE LOZANO JUSTE]

Does anyone know if this protocol works with regular BbsI instead of the Fast one? no need to phosphorylate annealed oligos? Do you think it can work with vectors containing BsaI instead of BbsI?

Thanks!!

[Bblues]

Hi, I tried this protocol with regular BBsI and it works fine. 

[SF]

you should not phosphorylate the oligos if you do the combined digestion/ligation because the restriction enzyme already leaves a phosphorylated end in the vector. if you phosphorylate the oligos, you have to cip (dephosphorylate) the vector and you cannot do the fast digestion/ligation protocol. 

BsaI is also a type IIS enzyme (cuts outside the recognition sequence), so my guess is that the ligation would be efficient as well- if the oligos are cloned in, it won't cut any more, but if the vector religates, it will cut again, giving you another chance to clone in the oligos. 

On Thursday, July 17, 2014 12:01:04 PM UTC-4, JORGE LOZANO JUSTE wrote:

[didier Fesquet]

Hi, I am surprised you use ligase buffer in your annealing step...95°C will damage ATP present in this buffer and interfere with ligation...
apparently, this is not true in your hands..

didier

[Phil Abbosh]

i think 95C is safe for ATP since PCR routinely gets that hot to melt and there is ATP in that reaction too.  I am not sure about DTT though.  It is in the NEB T4 ligase buffer.

[Anshuman Das]

Hi SF
I am using px334 vector. I followed the combined digestion ligation protocol using normal BbsI but I got almost equal number of colonies on ligation and self ligation plate. Is that normal to get so many colonies on self ligation?

[Peter Hohenstein]

Since BbsI digestions gives incompatible ends, you cannot get self-ligation (relegation of the tiny fragment you cut out is very very VERY unlikely due to the molar ratio). So if you have a lot of colonies on the self-ligation plate it is most likely the problem is with the digestion in the first place…

 

Peter

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[Phil Abbosh]

i had similar troubles on a negative control plate and just went with the two step procedure. i omitted the kinase step for the oligo insert but did gel purify the plasmid. its not that much more time consuming, and worked pretty reliably once i optimized the insert:backbone ratio.  using undiluted but annealed oligo (see earlier in this thread) did not work for me but it sounds like it works for others.  also, i still find that somehow my plasmid closes back on itself even though its not supposed to.  i found out afterwards that NEB's BbsI is supposed to be stored at -80C but i was keeping it at -20C.  i wonder if it damaged the enzyme and that is why i get plasmid closure.  i will post if my sequencing result is not correct in a few days.

[Vikram]

Hello Phil,

What was your observation after sequencing? Do you have any suggestions?

[Farshid Amiri]

?thanks a lot for your protocol. I have a question. you didn't add T4 ligase buffer

در پنجشنبه 17 ژوئیهٔ 2014، ساعت 3:01:55 (UTC+4:30)، SF نوشته: