Molecular Cloning Method Discussion

Problems with Quick change lightning site directed mutagenesis kit(STRATAGENE)

Thu, 19 Nov 2009 10:40:38 -0800

I had some problem about Site directed mutagenesis(SDM). Anybody can help me,please.

I used quik-change Lighting Site directed mutagenesis(SDM) kit to did mutant(single amino acid change).

This is my primers:

N164A _F 5'-ggacttatcactccctgggcttatcctctgctgatggc-3'
N164A _R 5'-gccatcagcagaggataagcccagggagtgataagtcc-3'

I used program of STRATAGENE to design primers:
http://www.stratagene.com/qcprimerdesign

Base length of each primers were 38 base and Tm of my primers were 78.97�C. The primers had GC terminate. The mutation is balanced as they are exactly in the middle. Everything is OK.

My plasmid+insert size is about 6.8 kb, I strictly follow the protocol. when I ran agarose gel after cut with DpnI, I saw many PCR product and its expect size correct.

But I don�t have any colony form transfromation of mutant into XL10-Gold ultracompetent cell. So I used my own completent DH5-alpha to did Heat-shock and I didn�t have any colony. I did Electrophoration using DH5-alpha for transfromation instread and this method I didn�t get colony again -*-

All method I added PCR product 2ul.

For control in this kit-pWhitescript- I did it already and it had strong band PCR and got many colony when transform into XL10-Gold ultracompetent cell.

I need to know which anybody has other efficiency transformation method to transform my product.

Anybody can help me,please.

Caren

Signal pIgplus vector sequence

Wed, 18 Nov 2009 09:54:46 -0800

Hi everyone,

I'm looking for the sequence of a discontinued vector once sold by R&D systems called Signal pIgplus. I'd be immensely grateful for any bit of sequence you could send me.

Cheers,
V

Promoter Cloning for Luciferase Assay

Tue, 17 Nov 2009 15:13:48 -0800

Hi,

I want to investigate a putative TF for a promoter with the Luciferase Assay. I am about to clone the promoter into the Luciferase vector. The +1 site of that promoter is followed by a 5'-UTR. My question is now, whether I should include downstream sequences and if yes how much of it (50, 100bp)? Or is it better, that my sequence stops at -1 directly followed by the luciferase gene.

To reformulate the question:
Does the region around +1 have any important sequence information for the transcription machinery?
Could it matter if I have additional 50-100bp upstream in the the luciferase assay in terms of the transcription of the gene or the stability of the mRNA? Does anybody have experience with that?

Thanks,

Manuel

Creating mutants

Mon, 16 Nov 2009 15:11:18 -0800

I have to create some double mutants to see how they function. I have created the single mutants in these combinations and cloned them to a plasmid. The size of the plasmid is ~11kb. In order to create the double mutants I would like to get some advice as to the best possible way to do it with minimal sequencing, and if possible eliminate the cloning process, (which I guess would not be easy!)

Adeno-x Tet-on vector problem

Mon, 16 Nov 2009 09:19:45 -0800

Hi everyone:

I am trying to ligate my GOI with adeno-xl Tet-on vector. It seems to be quite a tricky kit. I have constructed my recombinant shuttle-GOI plasmid and checked it with many different combinations of restriction digestions as well as PCR. All gave me desired results. However, when I cut this recombinant plasmid with I-Ceu and PI-Sce I, I got two bands 4.8kb and 3.4kb. However, I only expected the band sizes to be 2.8kb (the off-cut of the plasmid, should be the same size for all of us, right?) and 3.8kb (my GOI in the cassette). Surprisingly, if you add 4.8 and 3.4 is even bigger than 2.8+3.8. Isn't this ridiculous? Where could have this big plasmid ever been coming from? When I thought about this, I realized that I did not run the digestion product immediately, as my LD did not have SDS added. The two bands (4.8kb and 3.4kb) were given from the purified digestion product using the phenol-chloroform method same as manufacture's protocol. I then ligated the GOI fragment with adeno-x at 16C for 16hrs, cut with SwaI for 2hrs at 25C and transformed using DH5a, but on the next morning I observed nothing on the plate.

Do you have any idea of what might have happened according to your experience? Many thanks

Jarod

cloning protocol

Mon, 16 Nov 2009 06:05:19 -0800

Hi, can anybody tell me were I can get a protocol to clone pcr products with puc19 from fermentas_

Thanks

Ligation Time and Temp

Sat, 14 Nov 2009 08:23:40 -0800

Hello,

I amplifed my 700bp fragment with PCR and now I need to ligate a 700bp fragment to a 6kb vector using xhoI and NdeI sites. Would 90 min at RT or 90 min at 4C work?
Also, do i need to gel purify my pcr reaction before ligation? Will it work if i just use my pcr reaction directly or do pcr purify?

Thanks alot

Miniprep

Sat, 14 Nov 2009 08:13:35 -0800

Hello ALL,

For miniprep, the recommened incubation time is 12-16 hours and i was wondering if anyone has tried incubating for less time, such as 5-8 hours? Does less incubation time still give DNA?

THanks

PCR and then ligation

Sat, 14 Nov 2009 08:11:29 -0800

Hello,
I am doing PCR to amplify a 700bp region with primers with Tm= 81 and tm= 70 C. Would an annealing temperature of 65 work?

cloning problems in C. elegans expression plasmid

Fri, 13 Nov 2009 12:23:39 -0800

I am using the plasmid pPD49.83 from Andrew Fire kit. It has a heat shock
promoter in it and I am trying to put cDNA downstream of the promoter.
Also, I want to replace the promoter with my own promoters. But my
clonings are just not working. So, I wondered if you have faced similar
problems with using pPD vectors before. I am using NheI/SacI or NheI/NcoI
or SphI/BamHI for different clonings. Do you think some enzyme
combinations don't work well together?
Also, I was wondering if there is a Gateway system for C elegans
expression. Do you know about this? Are there any alternative/ easier
cloning systems like Gateway for C elegans?

3 way ligation help needed

Fri, 13 Nov 2009 12:13:06 -0800

I have 3 pieces of DNA that I need to ligate. They are all ~700 bp. The first piece has the start of gateway cloning site at the 5' end and the 3' end has a NotI site. The second piece has an NotI site at the 5' end and an XhoI site at the 3' end. The 3rd piece has an XhoI site at the 5' end and the start of the end of the gateway cloning site at the 3' end.

When I try to ligate the three pieces together I see a ladder on the gel with very few bands at the ~1800bp site. I have tried band stab PCR and have had no luck amplifying these bands (they could just be 3 random pieces ligated together). I have tried my ligations at RT for 10-30 min and the ligation always produces a ladder on the gel.

When I try to ligate only 2 of the piece I am having the same issue where I see a ladder.

Does anyone have any advice?

I cannot put the pieces directly into E. Coli yet because I need to run a round of PCR on the correct band to finish putting on the gateway cloning site.

I have thought about getting the TOPO kit and screening colonies but we do not have multichannel pippetts and am afraid that this will take forever since I have hardly any bands of the correct size. I am debating running the ligations on a gel and excising the correct band size (even if nothing is really shown) and trying to transform hoping the small amount of DNA that is there could get into the TOPO vector so I can replicate it in the bugs and do a colony PCR to amplify my region.

Mammalian expression vector for a partial protein fragment?

Fri, 13 Nov 2009 06:01:41 -0800

The whining continues I'm sort of hating my master's thesis as I'm facing a setback after a setback... but even if I never would work as a biologist after this, I want to survive and finish my thesis and get a new boss. So... by far:

1. I have managed to get the wanted protein fragment from the human cDNA library (the protein size is about 6000 bp, my fragment is from the c-terminal part and about 1000 bp)

2. I have cloned it into pET28 vector and expressed it in TOP10 and BL21(DE3) cells

I'm trying to purify the fragment now to sent it to the company... my goal is to get an antiserum against this protein's c-terminal part, which would stain tissues hopefully and which would at least change the world and give me a nobel price ok, maybe I'm just trying to finish the thesis at first

BUT, my thesis seem to be changing daily My supervisor wants me to express this insert in mammalian cells now and I'm trying to find a vector, which would include a signal sequence (as I only have a protein fragment, not the entire protein) and a his tag. He told me to just check out websites and find one (ignore the fact that two days ago he told me to do something else ), but by far I haven't found any information about is expressing partial protein possible in any of the vectors. So I have two questions...

1. Is this signal sequence normally included already in the vector?

2. Any suggestion about some good expression vector for a partial protein?

help with cloning using pBudce4.1

Fri, 13 Nov 2009 03:31:53 -0800

help....

hi,

i`m new to cloning, but i`ve read a lot about cloning from articles and textbook.
recently, i tried to clone a ~2kb insert into ~4.5kb pBudce4.1 from invitrogen.
the insert was amplified using primers with at least 6 nucleotides away
from the cutting site. i got a clear band at ~2kb from PCR using fermentas PhusionTaq.
then, the insert was eluted fromn gel and purified DNA conc was ok (~80ng/ul).
i used NheI and NotI to digest the eluted PCR product and i got clear band at ~2kb after gel check.
i also digested the pBudce4.1 using NheI and NotI, total volume 50ul and then eluted
the 3.5kb fragment. Gel check also showed a clear band at 3.5kb.

i tried to ligate this 3.5kb vector with the ~2.0kb insert. the molar ratio
i tried were 1:1, 1:3 and 1:5 using T4 DNA ligase (have tried both 16`c and rt O/N ).

plasmids were transformed into DH5a but there was no growth.
how can this be? my senior said that maybe the insert was not completely cut.
what can i do to improve the cutting?
tomorrow i will transform uncut plasmid to check the DH5a competency,
if the competency is ok, is there anything i could do to optimize the ligation condition?

before this, i have tried cloning into TOPO blunt end with the PCR products,
but i got no growth twice... pls help...

thank u in advance,
TY

Cloning and Expression Method Survey

Thu, 12 Nov 2009 18:28:00 -0800

Hi all,

I am trying to ask your opinions as I was trying to compare which is the best Expression kits/vector that you are using and if you would like to suggest and introduce others to use it, also your opinion about the PROS and CONS of the kits.

Thank you.

Bacteroides mutant construction

Thu, 12 Nov 2009 10:27:26 -0800

Hi
I'm wondering if I want to make a mutant in Bacteroides uisng SOE PCR. Would 800-1000bp fragment on each flanking side be enough for an efficient homologous recombination, like E.coli? Or it has to be around 2000-3000bp?
Thanks!!

Cloning woes - Running out of ideas!

Thu, 12 Nov 2009 08:14:58 -0800

OK. I am at my wit's end with this plasmid I've been attempting to construct. Here's what's going on in a nutshell:

Insert = 2775kb, cut from Promega pGEMt vector using BglII
Vector = 5100bp, cut with BglII and dephosphorylated with AP

After digestion, both insert and vector are visualized on a gel (next to uncut plasmids) and gel purified using Qiagen spin columns. The digestion does not seem to be an issue as I routinely get 2 bands when cutting the insert from the pGEMt vector and get 2 bands if I double digest the vector. Ligations (using a number of insert:vector rations) are carried out using NEB T4 ligase and are incubated overnight at 16C. Transformations are done using heat shock. I generally add 3-5 ul of ligation reaction to 25 or 50ul of cells. Cells are placed in SOC and allowed to recover for 1.5 hours before being plated on amp resistant plates. The positive control plate consistently has hundreds of colonies while the experimental plates have none - no background, nothing. I should also add that I have done the same procedure skipping the gel extraction step for my vector but still purifying it. I have done ethanol washes after the purifications just incase salt was carrying over (although I know this is not very likely considering I use Qiagen kits). And importantly, I have run a gel containing my ligation product in one lane, the same concentration of vector I added to my ligation in a second lane, and the same concentration of insert I added to the ligation in a third. In the ligation lane I did not see an 8kb fragment as expected. Instead, I saw a band for the vector, which was the same intensity as the just-vector lane, and a very faint band for the insert, which was much less intense with the just-insert lane. Hmmm...this last piece is puzzling. What's also puzzling is my lack of background colonies.

If you have any ideas, I'd really appreciate hearing them.

What fields of research other than microbiology work with bacteria and

Wed, 11 Nov 2009 20:03:47 -0800

I'm trying to get a research job but there aren't any labs dealing directly with bacteria in the area I want to work. Can anyone think of any other fields of research that may not study bacteria directly but still use bacteria and molecular cloning techniques in their research?

How to inoculate bacteria?

Wed, 11 Nov 2009 18:02:49 -0800

I'm having trouble because we use the 150ml? flasks to inoculate ecoli which for me are harder to inoculate than the 15ml tubes where I can twirl the loop to get bacteria off the end. But I'm not sure whether to use a pipet tip or use the loop to inoculate. When I use the tip, it doesn't always work and also when I used the loop it sort of works. The colonies I'm scraping from are very small which could be the problem. Anyhow I can't get the colony to come off the tip or the loop and into the liquid. Any suggestions? Also, what is the correct way of doing this because I know certain people have different preferences.

What retrovirus vetor can have 7.5kb insert

Wed, 11 Nov 2009 15:01:00 -0800

I am working on a large protein whose cDNA is 7.5kb big. I need subclone the cDNA into one retrovirus vector. Can anyone give some suggestions about retrovirus vectors that I can use?
Thanks in advance.

need help in my Fse1 restriction ezyme

Tue, 10 Nov 2009 03:39:44 -0800

hai..

could anyone help me in resolving this problem?
i had amplified Kan resistant gene with Fse1 restriction site(ggccggcc) at both end, I ligated in pGEMT vector and send for sequencing. Results from sequenceing showed that the both restiction site forward and reverse present. Thus i proceed with restriction enzyme Fse1 to cleave the Kan resistant gene in order to ligate it into my gene of interest (I use single digestion using Fse1 since that was the only option i had to carry out the cloning procedure). However after i treat the vector with restiction ezyme for 1hour and 3hours in 37C and run gel i could only observed a single band at 3800bases (pGEMT 3000bases+ Kan gene 800bases).

If im not mistaken the sigle band mean that the vector is in linearlized form, as compared to my contro(vector with untreated with restriction enzyme) would have 2 or more since it have supercoiled form.

Which mean that the restriction enzyme only act towards 1 site, but doesnt cleave at another site. Can anyone help me to solve this problem?y is it the ezyme doesnt cut at both site?What should i do?

protocol for RE

Vector-5ul
10Xbuffer 4-2ul
BSA-0.5ul
restiction enzyme-0.5ul
ddh2O-12ul
total-20ul