No bands on Colony PCR

[joshsimon]

Hey All

Am attempting colony PCR, yet no bands have shown up for either the wild type or mutant strain after two attempts. Are there common reasons or this occuring?

Interestingly, the reaction mix and PCR program had worked before, so am not sure why it's nto working now. Too many cells maybe?

Reaction mix was
150ul water
8ul of foward and reverse primers (1:10 dilution)
1ul of platinum taq polymerase
20ul buffer
6ul MgCl
2ul dNTP's (20umol/l)

Colonies were picked and added to 10ul milli q purified water. 1ul of this was added to 9ul of reaction mix.

PCR program featured 12 minute cell lysis phase at 94C, as well as 3m30s extension period at 68C (segment for amplification roughly 3.5kb)

Cheers

[mchlbrmn]

Your mix adds up to 187ul? If the buffer stock is 10x, I think it should be 180, so that's a little off. I suppose if the anneal temp were right at one of the primers' Tm, the more dilute salts could push the Tm over the edge so it couldn't anneal.
Primers and taq I don't know the concentration. OK, I looked up the Platinum protocol, and the taq looks right. Primers I don't know.
Mg is right if it's from 50mM as in the protocol.
dNTPs... if you mean 20 mM, OK.
I think a colony control is always a nice idea with a colony PCR. Unfortunately, I think this after I don't actually have good one on hand. If the PCR product is cloned from a different vector, bacteria containing that would be nice.
3.5 kb is a big to be able to readily PCR with standard taq, and a mix contaminated with bacteria. If it works routinely for you, than good, but it's possible you were fortunate the first time. Ideally you would have another internal primer on hand to PCR a smaller product.

[relaxin]

I agree with Mike that the PCR product of 3.5 kb is too big for Taq. If your primers are based on vector sequence, you can redesign another primer based on insert sequence around 500 bp from either ends. So if you use one primer based on vector sequence and another based on insert sequence, you can screen for clones with insert in the correct orientation.

The other reason for no bands on colony PCR is that none of the colonies you picked have insert. This is probably due to incomplete digestion and/or dephosphorylation of your vector.

[ashu330327]

Dear Sir
I am also not getting the exact band by doing colony pcr even with positive control. What could be the possible reason for this?
protocol
HPLC water 66 ul
10x buffer(15mM MgCl2) 10
MgCl2(25 mM) 5ul
dNTPs 2ul
FP(100pmole/ul) 5ul
RP 5ul
taq plymerase 2ul

Temp 4ul ---- one loop bacterial cells is innoculated in 100 ul TE n boil for 10 mins and centrifuge and supernatant is taken out.

result---
multiple bands of unspecific size were observed. Primers of inserts were used to check the recombinant clone in pk18mobsacB vector.
Thanks and waiting for your reply...

[mchlbrmn]

How big is the PCR product?
I use bacteria directly into the PCR reaction (which may be a bad idea), so in my hands the colony PCR is often messy. The bacteria often inhibits the PCR, and adds a smear of other stuff, so it works best for me when a small PCR product (=strong and efficient amplification) is amplified. This may require an internal primer inside the insert.

[relaxin]

Have you done PCR with the primers before? What is the size of amplicon?

I normally remove some bacteria from a colony using a filtered pipet tip and resuspend them directly into the PCR reaction mix by pipetting up and down. Alternatively, I use 1 ul of overnight culture. You resuspend a loopful of bacteria in 100 ul TE and take only 4 ul of the suspension. I wonder if there is enough of bacteria for the reaction. Can you see that the PCR mix becomes cloudy?

[ashu330327]

size of insert is 1.8Kb. i m not using 4ul of suspension but take supernatant so there is no qustion of cloudiness of pcr misture but ge tting the multiple bands which are not specific to this size. can i use the M13/PUC REV N FORWARD PRIMER TO CHECK THE RECOMBINANT PLASMID (PK18MOBSACB vector)

[relaxin]

If you put a loopful of bacteria in 100 ul of TE, centrifuge and use 4 ul of the supernatant for PCR, then you have no template. That explains why you have no band for PCR. The plasmid is in the bacteria, which you centrifuge down and discard. You should suspend the bacteria in 100 ul TE and take 4 ul of the suspension for PCR.

[ashu330327]

no sir I kept TEbuffer+loopful bacteria in boiling waterbath for 10 min and then centrifuge it for 3 mins at 8000rpm.

[ashu330327]

Sir please tell me what should i do according to your advice for doing colony pcr.

[relaxin]

Sorry, I forgot your boiling part. After boiling, the DNA should be released into the TE. I guess the problem may be not enough DNA template in the 4 ul TE.

I would just use a filtered pipet tip, pick up to half of the colony and suspend the bacteria directly into the PCR reagent mix. Pipet up and down and stir in the solution a few time to dislodge the hanging bacteria. You should be able to see a cloudy solution (this means there are bacteria in the reagent mix). At the initial denaturation step, the bacteria will be burst open and their DNA released for PCR to take place.

[ashu330327]

THANKS SIR!!!

[mchlbrmn]

I just noticed that you are using too much primer, if as written. You say you used 5 ul of a 100 uM stock in a 100ul reaction, so that comes to 5uM each primer. The normal PCR range is 0.2-1.0 uM concentration, with 0.5uM being a common concentration of primer to use. Too much primer can cause nonspecific bands, taking away reagents fromt he correct product. DNA (primers) also binds magnesium lowering the effective Mg concentration, which you are using a minimal amount of.

[JeanMarc]

ALTERNATIVE PROTOCOL :
You could use another protocol based on SDS-NaOH lysis of the colony bacteria (>=1mm diameter) and direct loading + migration on 0.8% agarose gel :
- prepare a 2X lysis solution :NaOH 0.1M /EDTA 10 mM / SDS 1% / Loading buffer 1x / 10% glycerol
- collect colonies on a p10 tip and put as much bacteria as possible at the bottom of an 1.5ml eppendorf tube. The tip could be added in parallel into 5ml selective medium for miniprep after ON culture at 37°.
- add 10 µl of water to eppendorf tubes, vortex, centrifuge briefly
- add 10 µl of 2x lysis buffer, let it 2-5 min at RT
- load into agarose gel wells not covered by TAE buffer (TAE buffer must be at half the height of the agarose gel, only to ensure electrical contacts because wells will leak). Let enter DNA into the gel with 10' of migration, cover the gel + TAE, migrate again.
- Good colonies containing recombinant plasmids will show a relative higher MW discrete band than non recombinant ones (since DNA was denatured the size will be different than expected, only shift in size is significant).

I could send you a typical image of such a gel if needed.
Best,

JM

[ashu330327]

dear sir
can I use m13_puc primers to check the inserts ligated in vector or not. I am using pk18mobsacB vector to construct deletion mutant of a gene. I am sending u a map of vector. I have cloned the insert in PstI site of this vector.