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Transformation keeps failing

shRNA cloning

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#1 inspiringMD

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Posted 08 February 2014 - 11:45 PM

Hi everyone!,

 

    I am an undergraduate student working as a research assistant and this is my first research experience so I am still adjusting to all of the techniques and procedures. My project is to clone shRNA into lentiviral vector (PLKO.1 TRC) and the basic protocol is as follows:

 

1. Anneal Oligos: mix, incubate at 95o C then let slowly cool down to room temperature.

 

2. Digest PLKO.1 TRC-cloning vector with Age1 and EcoR1

 

3. purify the 7kb band by gel extraction

 

4. Ligate using T4 DNA ligase: following 3:1 ratio of insert: vector (Note: my insert is 58 bp long), incubate at 16o C overnight.

 

5. Transform 4-5 μL of ligation mix to competent bacteria. My lab uses DH5-α E.Coli and Stable 3's. I tried transforming in both type of competent cells but still got no colonies!

 

Note: We use heat-shock method to transform (heat at 42o C for 45 secs then immediately on Ice for 2-3 min). Then the mix (ligation + comp cells) is recovered at 37o C in a shaking incubator.

 

I really don't understand where I keep going wrong and would like some help. My P.I. is unhappy with my inability to complete such a simple procedure.

 

What steps can I take to troubleshoot this and which points are critical? Thank you!

 

 

 



#2 perneseblue

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Posted 09 February 2014 - 12:13 AM

Okay.

First you need to confirm that the ligase is working. Run some of your ligated DNA onto a gel (use a narrow well). If the ligation worked, you should see high molecular weight bands, a smear larger than your 7kb vector.

 

The T4 ligase does inactivate over time. Is anyone else using this enzyme suffering ligation problems? The T4 ligase buffer will also degrade after repeated freeze thaw cycles (the ATP goes).  Does your ligase buffer still smell of DTT? If not, I strongly suggest you change buffer.

 

Second, please check to see if your annealed oligo insert actually forms overhangs that basepairs with your vector. Was there any mistake made in its design?

 

Third, did you dephosphorylate your vector? Unless specifically ordered, primer are not 5' phophorylated.


May your PCR products be long, your protocols short and your boss on holiday

#3 phage434

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Posted 09 February 2014 - 08:17 AM

Check that you are adding equimolar (not equal weight!) amounts of your insert and vector. You need to dilute your insert a great deal to make this happen. Your oligos should be annealed with some salt present -- perhaps 50-100 mM. Do the annealing prior to dilution, and then dilute into TE. Some easy things to check -- test the competency of your cells and your transformation technique. Transform serial 10x dilutions of your parent plasmid. You should get lots and lots  of colonies with 1 ng of plasmid, and hundreds with 10 pg.

This will also verify that you are using the correct antibiotic for your plates. I assume you are doing the recovery after transformation in non-selective medium. If not, you should be.

Another problem could be your gel purification. Exposure of gels to short wave UV can trash DNA. Use long wave, or better, blue light. Make very sure you have washed your columns well with buffer PE (or equivalent). Then make very sure you spin down in a dry collection tube at high speed to remove ethanol. Check that you really have DNA after the gel extraction -- run another gel if necessary.

Your ligation is done after about 10 minutes at room temperature. Only do it overnight if you are looking for an excuse to go home.







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