36 replies to this topic

[yean_ny_nie]

We use eppendorfs and they work very well the NYZ broth isnt a problem as we use SOC media (I dont know if this has something LB doesnt but maybe someone will!)

Good luck youll get there eventually!
[/quote]

Thanks Enthusiast for your reply. I will try using the SOC medium instead of LB broth. However, the XL-1 blue supercompetent cells are running out. Hence, I have to substitute it with self-prepared supercompetent JM109, which we have tested its transformation efficiency using pUC18. The plates shows good competency, however, when trying it (supercompetent JM109) with my dpn1 digested pcr product, it turns out no colony. I wonder what could went wrong.

Anyone could help with this as I have been repeating the procedure for quite some time. Thanks lots!!

[mastermi]

I prepare the sample reaction as below:
1ul of 10 reaction buffer
1ul (25ng) of 25ug/ml dsDNA template
1ul (125ng) of 125ug/ml forward primer
1ul (125ng) of 125ug/ml reverse primer
1ul of dNTP mix
Top up to 50ul with dH2O

The cycling parameter are as below:
i) 95oC for 30secs
ii) 95oC for 30secs
iii) 55oC for 1min
iv) 68oC for 1min/kb of plasmid length (mine would be 6mins)
(ii) to (iv) is for 16 cycles.

I wonder where goes wrong. Please help.

You really use just 1 µl of 10x reaction buffer for 50 µl reaction volume? Or did you just write it down false?
That would explain why you get no PCR product.
If you are using 5 µl, and the PCR doesn't work after several trials (annealing can be done at 50°-55°C), design new primers. Sometimes a pair of primers doesn't work for Quik Change for whatever reason...

[Kami23]

We use eppendorfs and they work very well the NYZ broth isnt a problem as we use SOC media (I dont know if this has something LB doesnt but maybe someone will!)

Good luck youll get there eventually!

Thanks Enthusiast for your reply. I will try using the SOC medium instead of LB broth. However, the XL-1 blue supercompetent cells are running out. Hence, I have to substitute it with self-prepared supercompetent JM109, which we have tested its transformation efficiency using pUC18. The plates shows good competency, however, when trying it (supercompetent JM109) with my dpn1 digested pcr product, it turns out no colony. I wonder what could went wrong.

Anyone could help with this as I have been repeating the procedure for quite some time. Thanks lots!!

The blue cells never ever work for us... I would try the ultracompetent XL gold cells! they are a bit more expensive but work every time (in my experience)

[yean_ny_nie]

I prepare the sample reaction as below:
1ul of 10 reaction buffer
1ul (25ng) of 25ug/ml dsDNA template
1ul (125ng) of 125ug/ml forward primer
1ul (125ng) of 125ug/ml reverse primer
1ul of dNTP mix
Top up to 50ul with dH2O

The cycling parameter are as below:
i) 95oC for 30secs
ii) 95oC for 30secs
iii) 55oC for 1min
iv) 68oC for 1min/kb of plasmid length (mine would be 6mins)
(ii) to (iv) is for 16 cycles.

I wonder where goes wrong. Please help.

You really use just 1 µl of 10x reaction buffer for 50 µl reaction volume? Or did you just write it down false?
That would explain why you get no PCR product.
If you are using 5 µl, and the PCR doesn't work after several trials (annealing can be done at 50°-55°C), design new primers. Sometimes a pair of primers doesn't work for Quik Change for whatever reason...

Opps, I'm sorry. I'm using 5ul instead of 1ul of the 10x reaction buffer. Thanks lots. I have design my primers for the second time already, however it turns out no results. Anyway, small and colourless colony were seen on the last batch of my transformation. Hopefully I can get something. And if not, I might lower down the annealing temperature as you said.

Thanks thanks.

[ah6tyfour]

I just did battle with a mutagenesis reaction and finally won after three weeks...so maybe I can be of some help.

I used Stratagene's QuikChange Lightning kit to mutate 5nt in each of three locations. But because the three locations are so close together, I had to do them one at a time (what a pain). First one went perfectly (300+ colonies), second one was okay (8 colonies...100% with mutation), third one didn't work. I tried four times and finally got it after I called Stratagene for troubleshooting.

Here's what they said:
-double extension time (so for Lightning kit, it's 30 sec per kb...do 1 min per kb instead)
-Use 8% final concentration of QuikSolution (normal is 1.5uL....use 4.08uL to get 8% in 51uL)
-Transform 4uL of reaction (their data shows no loss of efficiency up to 4uL)
-dNTPs might be bad

For me, I did everything they asked...and found the dNTPs were the issue. The protocol says to aliquot dNTPs to prevent freeze/thaw. So I put 1uL into each well of a strip tube. Problem is every time I had to do a reaction, I had to pull out the strip tube and cut off one of the tubes for my experiment. In the meantime, that 1uL thawed.

The tech support people do not know the formulation of the proprietary dNTPs, but know it is much higher than the usual 10mM. So it was suggested that I try using my own dNTPs and to try adding a lot more than I would for a normal PCR.

So I usually add 1uL of 10mM dNTPs for a 50uL PCR reaction. For the QuikChange reaction, I added 3uL of the 10mM.

I did two reactions identical in every way except for the dNTPs. In the end, the reaction with the kit's dNTPs had 0 colonies while my custom dNTP blend yielded about 20. 7 out of 8 clones I chose had the mutation.

I would try even more....maybe even 5-7uL.

[ah6tyfour]

From what you copy-and-pasted from the Stratagene primer design program, your primers have Tm 77-80 degrees C. You can use their Tm Calculator on the website to verify.

You'll probably find that the Tm is much higher than you think. Some of your primers are very GC-rich on one side even though the overall GC content is around 50%. This could result in your Sense and Antisense primers dimerizing. You need to INCREASE annealing temp, not decrease. Increasing will help decrease primer-primer interactions and also help increase specificity. Primer-primer interactions and loss of specificity will effectively take away most of your primers from participating in the reaction.

Try 60-degrees annealing. If that doesn't work, try 68-degrees. I always bump it up to 68 degrees because, if the reaction fails at 60 degree annealing, my first fix is to redo it at 68. So I soon realized I should just do them all at 68 to start with.

If you have the cool new ABI thermocycler, you could do both conditions at the same time =)

And lastly, check that you are calculating your primer ng/uL correctly. I always have trouble going from OD to ug. Good thing my primer company now includes a column on the data sheet that is simply "Total ug in tube".

Edited by ah6tyfour, 01 May 2009 - 10:59 PM.

[yean_ny_nie]

From what you copy-and-pasted from the Stratagene primer design program, your primers have Tm 77-80 degrees C. You can use their Tm Calculator on the website to verify.

You'll probably find that the Tm is much higher than you think. Some of your primers are very GC-rich on one side even though the overall GC content is around 50%. This could result in your Sense and Antisense primers dimerizing. You need to INCREASE annealing temp, not decrease. Increasing will help decrease primer-primer interactions and also help increase specificity. Primer-primer interactions and loss of specificity will effectively take away most of your primers from participating in the reaction.

Try 60-degrees annealing. If that doesn't work, try 68-degrees. I always bump it up to 68 degrees because, if the reaction fails at 60 degree annealing, my first fix is to redo it at 68. So I soon realized I should just do them all at 68 to start with.

If you have the cool new ABI thermocycler, you could do both conditions at the same time =)

And lastly, check that you are calculating your primer ng/uL correctly. I always have trouble going from OD to ug. Good thing my primer company now includes a column on the data sheet that is simply "Total ug in tube".

Thanks lots!! U've been very helpful.

[LucasRudy]

Hey all,

I'm as well struggeling with the site-directed mutagenesis. It turned out that 1 switched on the wrong PCR block after PCR reaction for the DpnI digestion and realized that after that one was finished so I added again 1µl DPnI inside and incubate the reaction eventually on 37 degrees.
I know it's a quite stupid error but I'm afraid now that my mutagenesis doens't work again although I tried it now for the fourth time.
Do you think this additional hour with DpnI on RT did affect my mutagenesis, although I incubate it afterwards once again with DpnI but this time on 37degrees?

cheers,
hoping to get any replies (please....."desparate")

[LucasRudy]

isn't that a classic one

[4leafclover]

Hi all

I've been having problems with the Stratgene's site directed kit too.. I've been trying to troubleshoot, and think its my primers 'coz I ran the control and it seems ok..WHY do primers for this kit have to be designed complementary to each other??? Previously whenever I'v designed primers they'v always been at two ends and never complementary.

So I'm wondering if primer dimers could be a problem. Currently my primers have a Tm of ~68 degrees. I do see primer dimer bands at the bottom of my gel and there is absolutely no amplification bands. Tried transformation, nothing. I've repeated this about 4 times now..

Would really appreciate some info.

[donny]

I've had some problems with SDM before too. I found that using less primers help. Either that reduces primer dimerisation or they don't consume dNTP too fast. Less template may help too, since template-PCR product hybrids are digested by DpnI too. I was told not to add too much dNTP 'cos there is a working stoichiometric range between Mg2+ and dNTP. I also used Phusion instead of Pfu since Phusion is much faster (15s/kb). But Phusion-amplified plasmids seems to have deletions near the ends of the primers.

[GNANA]

try to go with master mix, i have done successfully 8 mutations using Accuprime pfx supermix....u neednt hav to optimize anything here,,design primer as per the suggestion of stratagene and proceed, u ll get for sure if ur competent cells are good enough...get back to me if you need any more suggestions reg this....

good luck,
Gnana...

[janco]

hi, everybody, i also have a problem in using SDM. I planned to delete 6 nucleotides, so i designed the primers and performed the following steps according to SDM. Everything seemed OK and there were about 100 clones on the plate, so i picked 10 clones and sequenced after miniprep. But the sequence result shew that though the 6 nucleotides indeed deleted, there were only 70 nucleotides of my inserted sequence left (there should be 600 ) . So i cut the plasmid, and i found that the vector was only a little more than 3kb instead of original nearly 6kb.
Anyone ever had similar problem? I just got depressed by this strange result.

[GSUOC]

Hi,

You could have probably graduated by now. Anyways, my response to the above problem: Try not to mix the two primers in the initial thermal cycling. This worked for me. There could be a problem of the two primers attaching themselves.

Heat shocks must be timed very carefully and accurately to get efficient transformations.

good luck.

[Marite]

Hi all,

I just received the Stratagene's QuickChange kit and of course it didn't work....nothing new with using a new kit
Going down to troubleshooting my problem is the transformation. I even tried just the transformation control with pUC19 into XL-1 Blue cells and nothing not a single colony!!!

I proceeded exactly as the manual indicates:
Thawed the XL-1 Blue cells slowly on ice and aliquoted in 14 ml pre-chilled falcon BD tubes with round bottom.
Added 1 µl of pUC19 control vector, swirl and incubated on ice for 30 min.
Heat shock in a water bath at 42oC for 45 sec.
Incubated on ice for 2 min
Added 500 µl of NZY+ Broth pre-heated to 42oC
Incubated shacking for 1 h at 200 rpm: our shacker can not take any more rpm
Plated on Agar + ampicillin + X-gal + IPTG plates: 200 µl of NZY+ Broth and 5 µl of transformation control.
Absolutely no colonies the day after!!!
I just can't see what is going wrong in this little, basic and crutial part of the protocol.
Could anyone please help?
Thanks in advance