9 replies to this topic

[vetticus3]

Hi, and HELP!
I really need some advice on ligation.  This is the thing, I've managed to PCR a 300 bp product, using a forward primer (with an EcoRI site) and a reverse primer (BamHI site).  I'm purifying the product with QIAgen, double digesting it with EcoRI and BamHI...meanwhile, double digesting pBluescript KS+ also with EcoRI and BamHI....treating the cut pBSKS+ with CIAP... gel purifying it.... ligating the double digested 300 bp product into the cut pBSKS+ (about 50ng)  in a 3:1 molar ratio of insert: vector, using NEB T4 ligase and buffer, at room temperature (usually overnight, but at least 2 hours)... ethanol precipitating it, transforming it onto electrocompetent cells, and plating it O'night... blue white selection...controls of vector and ligase, and vector with no ligase... anyway, i'm lucky to get a positive colony on my test plates...  and with my one and only lovely positive colony, which I had pinned all my hopes on   , minipreped...double digested again using EcoRI and BamHI...ran an AGE gel to see if there was insert...and there was none, just bands at around 3000bp and higher.  (ok, occasionally i've gotten the insert, and then its party time...but really, it's getting ridiculous,)
I've repeated this again, and again.... I've used from 20 to 100 ng of vector, I've used 2:1, 3:1, 4:1, 100:1 ratios.  I even whispered sweet nothings into its ear.  
WHY IS THIS GOING SO WRONG??  I was thinking that the pBluescript is ligating to itself to make a "super big huge plasmid"... getting into the cells and appearing white.  But, to the point, why isn't the insert being shoved into the stinking little vector, and doing what it's supposed to, and why isn't this super huge big plasmid being shown on the gel when i was looking at how much background to expect??    with the ligations that i've run on the gel, it looks like there is no, and I mean no, background,  it's lovely and clean, nice band of cut vector, nice band of insert... absolutely nada in the region where the vector + insert in a circle should be.  
I've had it up to here with this, first the PCR wouldn't work for over a month because the bloody polymerase was dead/dying, the primers that were suggested to be used were stuffed, and the temperatures that were used were completely out of whack...finally get that to work, happy time   , cloning this should be a cakewalk, but it's not.  WHY ME??  
can anyone help me out?
I was thinking that since my PCR used primers with the EcoRI and BamHI site, maybe they don't need to be digested?  Maybe the ligation should be in a 37' water bath, or in the fridge, or spinning, or far, far away from me.  HELP!  
I really don't know what to do.  I will listen to *ANY* suggestions.
thanks,
vetticus

Edited by vetticus3, 28 September 2004 - 06:27 PM.

[pcrman]

Hi vetticus,

Quote

i'm lucky to get a positive colony on my test plates... and with my one and only lovely positive colony, which I had pinned all my hopes on  , minipreped...double digested again using EcoRI and BamHI...ran an AGE gel to see if there was insert...and there was none, just bands at around 3000bp and higher. no background, I was happy for a short while.

What is your positive control? You said you got colonies with positive control but there was no insert in it, right?

Quote

I was thinking that since my PCR used primers with the EcoRI and BamHI site, maybe they don't need to be digested?

No, you have to. I don't know how many extra nucleotides have you designed into the 5' end of your primers since restriction enzymes need some extra bases outside their recognition site, if there is not enough, your enzymes won't cut and ligation will fail.

[vetticus3]

My controls are: vector + ligase...which gives about 6 blue colonies; vector only, no ligase...which usually gives no colonies.  I've also used insert, no vector... no colonies.
No other controls.  I guess these are all negative, what should be used as a positive?  
I've got several 300 bp insert from different cell lines, and it's just not ligating.  
The point of ligating this is to get the insert sequenced.  If the bands from the PCR are really strong, is it really necessary to clone it?

My primers are 5'-GGGAATTCGGGACGAGAGGGCGACTT-3' for the EcoRI, and
5'-AAGGATCCCTCCCCTCTGGGCTACCT-3' for the BamHI.
I think they're ok.

Edited by vetticus3, 28 September 2004 - 06:43 PM.

[pcrman]

Yes, each primer has two extra bases outside the recognition site. Although only one extra is needed according to NEB sometimes two extra may just not enough.

Are the two enzymes compatible for double digestion?

Depending on the purpose of sequencing, usually there is no need to clone your PCR products before sequencing. Direct sequencing just does fine.

If you do want to clone it for seqeuncing, why don't you do a simple TA cloning which will save you lots of trouble.

Edited by pcrman, 28 September 2004 - 07:15 PM.

[vetticus3]

I'm looking for mutations in a stem-loop.  The idea is, or as i understand it, that in normal cells, the stem loop, just after the first intron,  acts as a stop sign to the polymerase.  When there's a mutation, the stem loop doesn't form, and the pol keeps on reading,  and ends up overamplifying the protein, causing all kinds of shinanigens... cancer mainly.  Thinking on it, it has be cloned... it's just such a huge pain in the butt.  
When i wasn't treating the pBSKS+ with CIAP, i was getting in excess of 10 000 colonies... but that was all background.  So il diluted it down, but it was still all background.  CIAP has at least stopped this, now it's just a matter of making it work.
The research officer in the lab (an angle) has used BamHI and EcoRI together, without any problems.  
Perhaps its just cursed.
TA cloning?  This is new to me... easier...hmmmm.  I'll try the ligation once more, and then try TA or witchcraft.  It has to turn out right one way or another.

Edited by vetticus3, 28 September 2004 - 08:23 PM.

[vetticus3]

Hello once again,
I've tried the ligation again, double digested insert, CIAP treated vector, 3:1 ratio....the bastard didn't work again.  I had one colony with insert on the whole friggen plate.  I give up.   This sucks like a hoover.
I'm thinking CIAP is CRAP.  When I wasn't going all fancy and phosphoryating the pBS, sure there was heaps and heaps of background, but there was RESULTS!!!! Sure i had to be really careful in picking the colonies....but hey it was worth it.  
Is it possible that my insert is making the vector toxic? and killing me, erghh ... i mean the bacteria?
I'm going back to what i know works.
Thanks for the advice, and letting me rant on a bit.
Cheers.
Vetticus

[SVTX]

Hey,

Easy way out.  I agree with pcrman.  Seems like you may not have to clone your PCR product.  One question needs to be clarified, how many bases away from the ends is your region of interest?  The first/last 30-40 bases of a template are never read by the sequencer.  More suggestions on "killer" cloning later!     

SV

[vetticus3]

It's a little over 70 in.  The sequencing is actually fine.
Hey, just wondering, is it possible to run a ligation out on a gel, cut out the band i want to stick in the bacteria, purify it, and then transform the cells?
I'm trying to be sneeky.
vetticus
in other news, completely screwed up my data presentation today....hahaha urg hh, whoever invented public speaking, i say this to you.... you bad!

Edited by vetticus3, 03 October 2004 - 08:48 PM.

[tfitzwater]

1. Too much CIAP will result in damaged ends on your vector due to exonuclease contamination in the CIAP.  Be sure that you add exactly the number of units that the supplier of your CIAP recommends for the number of ends you are dephosphorylating.  Be sure that you remove all traces of CIAP from the vector prep before use.  Phenol:chloroform extract twice.

2.  Switch to SAP to avoid this problem.

3. Cut out a BamH I/EcoR I fragment from another plasmid and confirm that you can ligatae that fragment into your vector and get plasmids with the right insert size.

4. Typically, you need at least 2x more colonies on the insert + vector ligation plate as on the vector only ligation plate to have a significant chance of getting your insert.  Presumably this is due to molecular crowding allowing better ligation of the vector to itself resulting from the presence of the insert in the reaction.

5. If the insert is toxic, grow the bacteria at 30C or lower.

[vetticus3]

my supervisor has said that the problem isn't the ligation, but the miniprep.  bad miniprep.  or that this isn't being digested properly, and that's why, when i run the gel, we can't see the insert.  it's being digest for 3 hours, what are you on about?  anyway, got a new miniprep kit,   ....there goes my weekend   .  
Also have to check for genomic DNA contamination.... 'cause one cell line specifically is going nuts.  (It' s making bands at around 15000 bp ).  So i'm transfecting with just that DNA, and ..yeah.
Will try another vector, thanks  .
My plates, untreated with CIAP look good today (~400 colonies on test, ~100 on controls).  will let you know in a couple of days how these turn out.

Thanks

vetticus

Edited by vetticus3, 07 October 2004 - 01:12 PM.