2 months in the lab now and can't get ANYTHING to work!!!

[MelissaThompson]

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So I joined this cancer research lab 2 months ago and can't seem to get ANYTHING to work right. I have made NO progress in these 2 months!!

Here is where my problem is (for all you bio-experts):

1) Cloning:

I get a good digest, confirm DNA after purification from gel, and after ligation & transformation I get very few colonies, of which NONE contain the insert.

I went back and tried many different things - altering the ratio of vector:insert, minimize UV, give the cells a longer recovery step...etc etc...STILL just very few colonies!!


2) siRNA transfection:

I repeated it about 100 times now, the cells NEVER respond efficiently!! On the western, it shows 80-90% knockdown, but the cells just don't show it phenotypically!!!



Other lab members are having GREAT success with these 2 procedures - but I am looking like an idiot, lab meeting after lab meeting, saying "I'm STILL troubleshooting"...


My main concern is now: will my PI fire me in the next month or so if I still can't get this to work? I received training to go over the procedures ONCE, and since then I have been on my own! The other grad student doesn't really want to help me 🙁

 

[AJgem]

what kind of e. coli cells are you using? there are cetain e. coli strains engineered to accept vectors better than others. You can ask your PI to order the better strain if you don't have it...also molecular biology is like voodoo magic! 2 months is actually not to long just continue to troubleshoot!

also, what phenotype are you looking for? Is this a cell specific knock-down or global? If its not targeted to a specific cell and you do not care about this then the western should be verification enough...also you might not be affecting the mRNA level (only protein) and that is why you see the reduced expression from the western but the phenotype effect is at the mRNA level...

 

[MelissaThompson]

what kind of e. coli cells are you using? there are cetain e. coli strains engineered to accept vectors better than others. You can ask your PI to order the better strain if you don't have it...also molecular biology is like voodoo magic! 2 months is actually not to long just continue to troubleshoot!

Commericially made competent DH5-alpha cells.


I know the problem is not with the reagents - because other lab members are having GREAT success doing this using the same reagent/material - its somewhere in my technique/procedure that I'm not aware of!

 

[AJgem]

technique- vortex everything! it really does matter

 

[jkm07]

1) Cloning:

I get a good digest, confirm DNA after purification from gel, and after ligation & transformation I get very few colonies, of which NONE contain the insert.

I went back and tried many different things - altering the ratio of vector:insert, minimize UV, give the cells a longer recovery step...etc etc...STILL just very few colonies!!

Is this a single restriction enzyme digest or a double RE digest? Single RE digests are a pain because of self-ligation. Do you use alkaline phosphatase after the digestion step? That can help with preventing self-ligation. You can also let the ligation reaction sit for a longer time.

Keep your head up! It happens to the best of us. I've had my share of SNAFUs in lab.

EDIT: Also one more thing, rather than altering the ratio of V:I, you can double up on the amount of DNA in the reaction to about 50-100 ng. In the past, my ligation reactions weren't working because there just wasn't enough DNA, though the ratio was around 1:3/1:5

 

[jkm07]

technique- vortex everything! it really does matter

Solid advice 👍

 

[AJgem]

Is this a single restriction enzyme digest or a double RE digest? Single RE digests are a pain because of self-ligation. Do you use alkaline phosphatase after the digestion step? That can help with preventing self-ligation. You can also let the ligation reaction sit for a longer time.

Keep your head up! It happens to the best of us. I've had my share of SNAFUs in lab.

EDIT: Also one more thing, rather than altering the ratio of V:I, you can double up on the amount of DNA in the reaction to about 50-100 ng. In the past, my ligation reactions weren't working because there just wasn't enough DNA, though the ratio was around 1:3/1:5

also, do a PCR purification on your PCR products after you CIP it!

 

[clownbabyMD]

Commericially made competent DH5-alpha cells.


I know the problem is not with the reagents - because other lab members are having GREAT success doing this using the same reagent/material - its somewhere in my technique/procedure that I'm not aware of!

Do you inactivate your ligase, following the ligation? I typically place my ligation reaction at 65C to inactivate. The leftover ligase can inhibit downstream transformation steps.

Also, if you're having trouble cloning in general, you can always try "sub-cloning". You can use a super dependable vector like pGemT (made by Promega) for a multistep cloning process. If you use this, you can directly ligate your PCR product into the vector, and then cut it out of pGem, finally ligating into your final vector of interest. I know this sounds round-about, but it seems to drastically improve my chances of successful cloning.

I definitely know how you're feeling. I went through the exact same frustrations. Don't get down on yourself, because even though you might be carrying out each step perfectly, the "voodoo" of cloning will no doubt get the best of you sometimes. Good luck!!

 

[Arbor Vitae]

So I joined this cancer research lab 2 months ago and can't seem to get ANYTHING to work right. I have made NO progress in these 2 months!!

Here is where my problem is (for all you bio-experts):

1) Cloning:

I get a good digest, confirm DNA after purification from gel, and after ligation & transformation I get very few colonies, of which NONE contain the insert.

I went back and tried many different things - altering the ratio of vector:insert, minimize UV, give the cells a longer recovery step...etc etc...STILL just very few colonies!!


2) siRNA transfection:

I repeated it about 100 times now, the cells NEVER respond efficiently!! On the western, it shows 80-90% knockdown, but the cells just don't show it phenotypically!!!



Other lab members are having GREAT success with these 2 procedures - but I am looking like an idiot, lab meeting after lab meeting, saying "I'm STILL troubleshooting"...


My main concern is now: will my PI fire me in the next month or so if I still can't get this to work? I received training to go over the procedures ONCE, and since then I have been on my own! The other grad student doesn't really want to help me 🙁

1. Cloning can be hard, and takes a while to master. Don't sweat it. Two months is nothing.


2. What are you trying to knock down with your siRNA? Maybe it just doesn't show phenotype, since you are already confirming with western blot? Also, I do something similar with siRNA and I have to wait an additional 48-72 hours after treating and after peak knockdown to see any phenotype. Basically my cells have to go through a couple of cycles to show a change. You may even have to treat a few days in a row.

 

[EBTrailRunner]

Technique, technique, technique. Have a more experienced lab member watch you from start to finish and offer any advice.

 

[btown8908]

I agree with the others: Technique is so important.

Transfection can be tricky and I suspect it boils down to how you shake your plates or pipette reagents. Are you using Lipofectamine by chance? If you're using tough to transfect cells, this can be a issue.

Paying attention to the smallest of details really improves your results. Also, make sure you're using the right reagents haha.

Have someone watch you to make sure you're doing it the right way.

Good luck!

 

[saveourpens]

Assuming your lab practice is fine, most research is trying to understand failures anyway.

 

[1TB4RKSB4CK]

If your lab has sufficient grants/funds etc. Look up variations of the protocols you are doing.

 

[deleted357625]

wut duh fack?

 

[naomisky]

Sorry about your troubles! cloning is massively hard until you get into the groove.

What kinds of controls are you performing? can you try to transform your DH5a's with a control plasmid, like a pUC19 or something? maybe it's a bad batch of cells.

I have found that during the transformation process, the cells are sensitive to the actual volume of DNA added, not just the mass. for 50ul of cells (I use invitrogen's Top 10 competent cells) 2ul of DNA is optimal in my experiments...not more, not less. I've transformed with volumes ranging from 0.5ul to 5ul.

Also, check out Sambrook's Molecular Cloning for a sample protocol. Feel free to P.M. me if you want me to send you my adaptation of this protocol.
good luck to you (because luck trumps skill in cloning..)!