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Sequencing problem and restricition enzyme digestion


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#1 helpwithdna

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Posted 07 May 2013 - 12:11 PM

Hi all,
I currently receive my sequencing result but i found some problems with it!
My work:
get dna sample -> amplified desired sequence by our primers -> run gel to confirm whether the product is correct or not -> if correct, go ahead with Restriction enzyme digestion (plasmid and pcr product) -> run gel -> if correct, go ahead with ligation -> transformtion -> pick colonies -> purify -> RE again (confirm whether the plasmid take up the DNA) -> sequencing

My target sequence should be around 500b.p. However, after ligation, I do the transformation and restriction enzyme digestion and then finally agarose gel electrophoresis again, the bands shown were not at 500b.p. Bands at different position (low M.W. and High M.w.) were shown.

Is there something contaminate the sample?

On the other hand, I would like to ask what is the ideal concentration and OD ratio for plasmid? Thank you!

Edited by helpwithdna, 07 May 2013 - 12:55 PM.


#2 helpwithdna

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Posted 07 May 2013 - 12:58 PM

Hi all,
I currently receive my sequencing result but i found some problems with it!
My work:
get dna sample -> amplified desired sequence by our primers -> run gel to confirm whether the product is correct or not -> if correct, go ahead with Restriction enzyme digestion (plasmid and pcr product) -> run gel -> if correct, go ahead with ligation -> transformtion -> pick colonies -> purify -> RE again (confirm whether the plasmid take up the DNA) -> sequencing

In my ligation, i tried out the 1:1 , 3:1 ratio of insert to vector.
The competent cells were prepared and used by all lab groups.

However, after transformation, we have found little (1:1) or actually no colonies (3:1) for most of the plates.
I pick up one of the only white colonies and uses it for later restriction enzyme digestion process.
The result of gel electrophoresis was not good. My target sequence should be around 500b.p. The bands shown were not at 500b.p. Bands at different position (low M.W. and High M.w.) were shown.

Is there something wrong with our ligation and transformation technique?
On the other hand, I would like to ask what is the ideal concentration and OD ratio for plasmid? Thank you!

#3 phage434

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Posted 07 May 2013 - 03:46 PM

You can sequence directly from your PCR product. Purify the product and add each of the two primers separately. With a 500 bp fragment, you will likely get complete coverage.

The problem you have could be in many places. Are there 5' bases outside of your RE cut site on the primers? Do you purify the PCR product prior to the RE digest?

#4 christy

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Posted 07 May 2013 - 03:53 PM

Hi,

What is your plasmid? Is there any restriction site what you are using in the plasmid or insert? What are the enzymes? Some restriction enzymes have star activity, if you incubate long time that will chop the DNA.

#5 helpwithdna

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Posted 07 May 2013 - 09:59 PM

Due to time-constraint and limited resources, I cannot send out my direct PCR product to sequencing. However, I would like to find out why my sequencing result is false. I have purified my product before the last RE. In my first RE, the gel electrophoresis shows that my PCR and Plasmid were cut correctly.

My plasmid is p-GEM-3z. I have blast the sequencing results. I have chosen the most similar human gene for analysis. I found the restriction sites used (bamh1 and ecor1) on that gene. However, the size of this insert (~1000b.p.) does not match the last gel electrophoresis result(where band was found at ~1500b.p.)

The last restriction enzyme, due to some errors, it only incubated for around 2 and a half hour, originally 4 hours were needed.

Thank you for your help.

Edited by helpwithdna, 07 May 2013 - 10:01 PM.


#6 christy

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Posted 07 May 2013 - 10:22 PM

Hi,

I think after sequencing your insert you can figure out. If the restriction site bases are wrong you may not get your target gene after digestion. The enzyme added to the reaction may cut the enzyme present in the pGEMT vector.

#7 christy

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Posted 07 May 2013 - 10:23 PM

Enzyme Cat# Temp Supplied NEBuffer Supplements % Activity in NEBuffer BSA SAM 1 2 3 4 BamHI* R0136 37°C NEBuffer 3 Yes No 75 100 100 100 EcoRI* R0101 37°C NEBuffer EcoRI No No 100 100 100 100
Double Digest Recommendation(s) for BamHI + EcoRI:

  • Digest inNEBuffer EcoRI + BSA at 37°C.
    Note: BamHI may exhibit star activity in this buffer.
Note: The above recommendation is based on the experimental results. Please checkSuggested NEBuffers for Double Digestion.
* BamHI has a High Fidelity version (R3136)
EcoRI has a High Fidelity version (R3101)
High Fidelity (HF) Restriction Enzymes have been engineered for reduced star activity
and have 100% activity in buffer 4 which may simplify your double digest.

#8 helpwithdna

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Posted 07 May 2013 - 11:28 PM

Hi,

I think after sequencing your insert you can figure out. If the restriction site bases are wrong you may not get your target gene after digestion. The enzyme added to the reaction may cut the enzyme present in the pGEMT vector.


The control A sequence from the sequencing result is the p-GEM vector.
In the T7 promoter sequence, I blast the results and it is not similar with the pGEM vector
In my colonies, both white and colonies were found... Why would this happen??

#9 helpwithdna

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Posted 07 May 2013 - 11:29 PM

Enzyme Cat# Temp Supplied NEBuffer Supplements % Activity in NEBuffer BSA SAM 1 2 3 4 BamHI* R0136 37°C NEBuffer 3 Yes No 75 100 100 100 EcoRI* R0101 37°C NEBuffer EcoRI No No 100 100 100 100
Double Digest Recommendation(s) for BamHI + EcoRI:

  • Digest inNEBuffer EcoRI + BSA at 37°C.
    Note: BamHI may exhibit star activity in this buffer.
Note: The above recommendation is based on the experimental results. Please checkSuggested NEBuffers for Double Digestion.
* BamHI has a High Fidelity version (R3136)
EcoRI has a High Fidelity version (R3101)
High Fidelity (HF) Restriction Enzymes have been engineered for reduced star activity
and have 100% activity in buffer 4 which may simplify your double digest.


Thank you for your information. I initially tended to digest for 4hrs at 37 degree Celsius. However, due to some errors, it only digested for 2 hours. will this lead to the results i described? (many differ M.w. bands on my gel). Thank you

#10 phage434

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Posted 13 May 2013 - 04:48 AM

Most digestions are finished in 5-15 minutes. I never digest longer than 1 hour, often for 30 minutes. This should have zero effect on your sequencing.




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