Molecular Biology Troubleshooting Forum

Molecular Biology Troubleshooting Forum http://www.protocol-online.org/forums Fri, 10 Feb 2012 11:41:32 +0000 60 Help. Lentivirus. Good titer but no transduction http://www.protocol-online.org/forums/topic/24371-help-lentivirus-good-titer-but-no-transduction/
Anybody can help me????????? Please I do not what can I do.]]> Fri, 10 Feb 2012 11:41:32 +0000 http://www.protocol-online.org/forums/topic/24371-help-lentivirus-good-titer-but-no-transduction/ DNA Shuffling http://www.protocol-online.org/forums/topic/24369-dna-shuffling/
I'm working since last month with a DNA shuffling strategy to improve the enzimatic activity of 1 target enzyme.

I follow the original protocol developed from Stemmer in 1994, but I have many problems after Primerless PCR.

I always get an amazing smear of big size. I use different dilutions of this product to performe the second PCR but I've never got the right size band (2.300 kb).

Does anybody have experience with this technique? May you give me some advice?

Thanks,
RV]]> Fri, 10 Feb 2012 09:30:56 +0000 http://www.protocol-online.org/forums/topic/24369-dna-shuffling/ Weight of a single Lambda DNA. http://www.protocol-online.org/forums/topic/24361-weight-of-a-single-lambda-dna/ Can anyone please tell me the weight of a single lambda-DNA molecule of 24000 bp? Thu, 09 Feb 2012 20:11:51 +0000 http://www.protocol-online.org/forums/topic/24361-weight-of-a-single-lambda-dna/ RBS site removed in construct. Will there be expression? http://www.protocol-online.org/forums/topic/24358-rbs-site-removed-in-construct-will-there-be-expression/

Thanks]]> Thu, 09 Feb 2012 18:37:19 +0000 http://www.protocol-online.org/forums/topic/24358-rbs-site-removed-in-construct-will-there-be-expression/ DEPC treatment (EDTA - AcONa) http://www.protocol-online.org/forums/topic/24354-depc-treatment-edta-acona/ And Na Acetate?

Hope you can help me!!]]> Thu, 09 Feb 2012 14:56:10 +0000 http://www.protocol-online.org/forums/topic/24354-depc-treatment-edta-acona/ plasmid extraction http://www.protocol-online.org/forums/topic/24341-plasmid-extraction/
10 ml culture cell + kanamycin overnight at 37C
observe grow or not
transfer into falcon tube, centrifuge 13000 rpm, at 4C, 10 min
remove supernatant
add 100ul alkaline lysis solution 1, mix the pellet
transfer into apendorf tube
put on ice
add 200 ul solution 2,invert 6 times, put on ice 3-5 min
add 200 ul solution 3, invert
centrifuge 5 min, 4C, 13ooo rpm
transfer supernatant into new tube
add 5 ul RNAse, put in water bath , 10 min at 37C
add phenol ,same volume with supernatant
centrifuge 5 min
transfer the upper layer into new tube
add chloroform , centrifuge, transfer the clear solution into fresh tube (wash 3 times with chloroform)
add 2 volume of alcohol 95%, centrifuge, remove supernatant
add 70%alcohol , centrifuge
remove supernatant, dry overnight
add eluent buffer, store at -21C]]> Wed, 08 Feb 2012 15:58:18 +0000 http://www.protocol-online.org/forums/topic/24341-plasmid-extraction/ BAC library screening using PCR http://www.protocol-online.org/forums/topic/24339-bac-library-screening-using-pcr/ Hello,

I am currently [color=#0066FF !important]working[/color][color=#000000] on a project where we have received a BAC [/color][color=#000000]library[/color][color=#000000] with 100 clones pooled per well. My boss has said we will screen using PCR. We will do an initial screen with PCR, then for the wells which received a postitve reaction we will dilute then 1/96 and PCR again. We will then sequences using 454 the diluted [/color][color=#0066FF !important]positive[/color][color=#000000] wells. I cannot find any material online about this, and I am not sure if my supervisor knows an exact protocol. I am slightly confused about this whole method, is there anyone who can further explain it to me or provide me with a reference on this???[/color]

The only information I find is about superpooling and 3D plates etc. We are not using this, we only have the plates of 100 clones pooled per well.

[color=#000000]Any input is greatly appreciated, thank you.[/color]]]> Wed, 08 Feb 2012 15:04:19 +0000 http://www.protocol-online.org/forums/topic/24339-bac-library-screening-using-pcr/ ligation colonies contain empty vector.. http://www.protocol-online.org/forums/topic/24336-ligation-colonies-contain-empty-vector/
I was wondering if anyone had any advice! I am trying to subclone a 4.2kb insert into 5.2 vector..... I am cutting with eco and bam.. i digested and ran on a gel and purified the insert and vector from the gel. following ligation and transformation there are no positive clones.

Now, the negative control always has colonies.. I know you can only test if the vector was single cut by each enzyme separately and assume the double worked. So I had the same vector with an insert in it already and cut that with eco and bam and I could see the insert dropout. I then took the backbone of that and one would assume that this dna had to be double digested if the insert dropped out. so why is there colonies on the negative control?

Please help!!!]]> Wed, 08 Feb 2012 13:11:15 +0000 http://www.protocol-online.org/forums/topic/24336-ligation-colonies-contain-empty-vector/ cDNA or genomic DNA for over expression in Arabidopsis http://www.protocol-online.org/forums/topic/24313-cdna-or-genomic-dna-for-over-expression-in-arabidopsis/ I have to over express quite a few genes in Arabidopsis. I am planning to go ahead with cDNA of gene. But I am little worried if I will be in trouble because some genes has some regulatory sequences in introns as well. So is it safe to use genomic DNA? Anybody faced any problem by using cDNA ? Please let me know if you can help with some suggestions. Thanks in advance.....]]> Tue, 07 Feb 2012 09:21:19 +0000 http://www.protocol-online.org/forums/topic/24313-cdna-or-genomic-dna-for-over-expression-in-arabidopsis/ <![CDATA[RNA isolation- can't get pellet after isopropyl]]> http://www.protocol-online.org/forums/topic/24310-rna-isolation-cant-get-pellet-after-isopropyl/
It was my first time doing RNA isolation.

]]> Mon, 06 Feb 2012 21:54:26 +0000 http://www.protocol-online.org/forums/topic/24310-rna-isolation-cant-get-pellet-after-isopropyl/ Reverse Transcription PCR Problem with Primer pair http://www.protocol-online.org/forums/topic/24309-reverse-transcription-pcr-problem-with-primer-pair/ I started recently to study the co-transcription of some catabolic genes in a gram positive bacterium with Reverse-Transcription PCR. The primer pairs which i designed seems to work good though the bands are not very intense.
Before i use the primers in RT-PCR i checked them in genomic DNA and all of them gave the desirible products.The problem is one pair which although gives one band with genomic DNA, in RT-PCR gives two bands.
I note that i used RobusT RT-PCR kit for Reverse-Transcription.
If anyone has an idea for what is happening, i would like to know...

Thanks!!]]> Mon, 06 Feb 2012 20:45:39 +0000 http://www.protocol-online.org/forums/topic/24309-reverse-transcription-pcr-problem-with-primer-pair/ High RNase concentration interfering with PCR? http://www.protocol-online.org/forums/topic/24298-high-rnase-concentration-interfering-with-pcr/
I started working in a new lab last week and their protocols seem a little bit awkward to me.
Referring to a DNA extraction protocol from cherry fruit flies:
After they extracted and precipitated the DNA and dried it in the SpeedVac, they add TE buffer + RNase and let it dissolve over night at room temperature. I was told to add RNase A to an end concentration of 10mg/ml in the buffer. In my opinion this seems to be a little bit to high? I am afraid that it could interfere with the PCR reaction.

In fact the PCR reactions did not work, I used older DNA sample they gave me and samples I isolated by myself. However, I used a lambda test kit in parallel, which worked perfectly fine. So I am thinking that either the primers (although I tried several ones) might be to old (they told me that they successfully used the same primers in the past with the same PCR program) or there is really a problem with an too high concentrated RNase.

I would really appreciate it if you have some ideas for me!

Best regards,
Ikar]]> Sun, 05 Feb 2012 17:00:01 +0000 http://www.protocol-online.org/forums/topic/24298-high-rnase-concentration-interfering-with-pcr/ SNP and SNV http://www.protocol-online.org/forums/topic/24286-snp-and-snv/
Hi there,
I thought that SNP is a single nucleotide polymorphism - which happens among a population, whereas SNV is a single nucleotide variation among an individual. So SNP should contain more alleles... Can someone please verify the answer and if it's wrong, explain why?
Thank you!]]> Sat, 04 Feb 2012 08:15:06 +0000 http://www.protocol-online.org/forums/topic/24286-snp-and-snv/ failure of PCR from yeast genomic DNA http://www.protocol-online.org/forums/topic/24279-failure-of-pcr-from-yeast-genomic-dna/
I got a problem here... Plz help... Posted Image

I tried to delete a gene in baker's yeast by homologous recombination and need to confirm the gene replacement by PCR. I transformed yeast with the deletion cassette and a couple of candidates showed up and I picked up 6 to test for deletion.
Our group always isolate genomic DNA and do PCR to test. Normally I grow cell in rich medium YPD to isolate the genomic DNA and it worked for me almost every time.
But this time there is a plasmid I need to maintain so I grew candidate cells in selective minimal medium. I used 5ml overnight minimal medium culture for genomic DNA isolation (phenol/chloroform and glass beads vortex method), everything looked fine - I got some white DNA pellets and washed once with 70% ethanol and then dissolved in dd water.
Then I did PCR on these obtained 6 genomic DNAs along with a positive control (WT genomic DNA isolated from YPD culture). Weird thing is, the positive control showed a nice band at right size but no any band showed up for all my 6 candidate genomic DNA!!

This is the second time I observe this problem. Last time I had the same problem but after growing cells in YPD I got successful results. But this time, as I mentioned, I need selective minimal medium to maintain a plasmid (with essential gene on it).

I was careful to make sure no phenol phase was transferred, so it should not be phenol problem. I should have isolated enough genomic DNA as I could see a good amount of DNA white pellet after ethanol precipitation.

Anybody had similar experience? Probably some Taq inhibitors were carried over (somehow not a problem for YPD culture)? If so, what is the best way to remove them?

Thanks in advance.]]> Fri, 03 Feb 2012 19:09:24 +0000 http://www.protocol-online.org/forums/topic/24279-failure-of-pcr-from-yeast-genomic-dna/ troubles in DNA digestion http://www.protocol-online.org/forums/topic/24272-troubles-in-dna-digestion/ I've been trying to digest pGEX-4T-1(4kb) and DNA sequence of Mycoplasma (514pb) using SalI and EcoRI for 4 weeks.. But i can find nothing after runnig an agarose gel (1%).
I know that 1U of the enzyme digest completly 1µg of DNA in one hour at 37°C. but I'm not able to exploit this knowledge

I'm using the following protocol;

H2O 5µl
OPA buffer 2x 1µl
DNA / pGEX 4µl
SalI 15U/µl 0.01µl
EcoRI15U/µl 0.01µl

I tryed diffrent times of incubation at 37°C: 60; 45 and 30 minutes.
I changed diifrent volumes of DNA.
but i still find nothing on agarose gel Posted Image

questions are:

1- How can i know if my enzymes are ok (as they ar not new)?
2- as i can't know the concentration of my plasmide neither my DNA, how can i calculate the number of unit of enzyme i'm supposed to use?

3- How can i estimate the concentration of DNA after running an agarose gel?
Has anybody had the same problems or do you have an idea what i'm doing wrong ?

( in the pic of agarose gel, i put 3 µl of pGEX as positif control : non digested plasmid)
If any one can help, I'll be really grateful Posted Image

Attached thumbnail(s)

]]> Fri, 03 Feb 2012 11:03:52 +0000 http://www.protocol-online.org/forums/topic/24272-troubles-in-dna-digestion/ Best plasmid for GFP + Mammalian Selection http://www.protocol-online.org/forums/topic/24270-best-plasmid-for-gfp-mammalian-selection/
I plan on performing a nucleofection on primary neural progenitor cells derived from IPSCs so I can label them with GFP and generate a stable line. The only vectors I have found that are compatible are for lentiviral transductions such as this one: http://www.addgene.org/browse/sequence/8617/

I plan on linearizing the GFP + puromycin region then performing the transfection. Do I need to add in PolyA signals downstream of GFP and puromycin in order to get expression to generate stable clones? Do any of you know of any other plasmids on addgene which would have GFP + a selectable marker that are not designed for lentiviral work?

Thanks]]> Fri, 03 Feb 2012 04:05:49 +0000 http://www.protocol-online.org/forums/topic/24270-best-plasmid-for-gfp-mammalian-selection/ Transformation /Cloning Problem http://www.protocol-online.org/forums/topic/24264-transformation-cloning-problem/
I have been working for already a few weeks on my transformation/cloning reaction. However up till today with no results :-(

I have been trying to ligate a 1,5 kbp insert into a 8 kbp vector.

First of all I digest my vector and insert with Not1, afterwards I gel purify both.
Then I dephosphorylate my vector with CIP and gel purify this again.
Followed by a quick ligation (NEB) reaction with a 1:3 vector/insert ratio (5min at RT).
Afterwards I transform my SURE2 cells with 1 ul of the ligation reaction (in 10ul competent cells).

My positive control gave me good results however no colonies were seen on my experimental plates....

Can somebody please help me with this?

Thanks,

Alex

PS I have the intention that I lose quite a lot of product after gel purification... However I still have enough to start up a 3:1 ligation reaction...]]> Thu, 02 Feb 2012 20:51:19 +0000 http://www.protocol-online.org/forums/topic/24264-transformation-cloning-problem/ Ligation Problem http://www.protocol-online.org/forums/topic/24262-ligation-problem/
I am recently having problems in ligating an insert into a vector. My vector is a 8kbp construct and my insert about 1,5 kbp. However after dephosphorylating my vector i have no colonies at all (Using T4 dna ligase). So I switched to the quick ligase kit, and they recommend to use 50ng of the vector and a 1:3 ratio for each insert. But is 50ng enough? As my vector is quite large, I am not sure whether 50ng of vector would be sufficient for the ligation reaction...

Thanks,

Alex]]> Thu, 02 Feb 2012 20:19:42 +0000 http://www.protocol-online.org/forums/topic/24262-ligation-problem/ a robust multi-gene cloning method http://www.protocol-online.org/forums/topic/24252-a-robust-multi-gene-cloning-method/ http://dx.plos.org/10.1371/journal.pone.0030267
please return your comments.]]> Thu, 02 Feb 2012 07:47:52 +0000 http://www.protocol-online.org/forums/topic/24252-a-robust-multi-gene-cloning-method/ <![CDATA[I've run out of ideas.... Restriction enzyme digestion]]> http://www.protocol-online.org/forums/topic/24238-ive-run-out-of-ideas-restriction-enzyme-digestion/
Does concentrated TE cause RE cutting problems?]]> Wed, 01 Feb 2012 02:36:00 +0000 http://www.protocol-online.org/forums/topic/24238-ive-run-out-of-ideas-restriction-enzyme-digestion/