Tal Lab
From WittleNet
Contents |
Inroduction
Lab members come and go and when they go, sometimes valuable information is lost. This is an effort to retain that knowledge in the form of protocols, and most importantly, people's personal tips and tricks. Where are the crucial steps in a protocol and how do you deal with them? Please post your protocols, your tips, your links etc. here. Post all the little things that seem obvious too. This should be a complete resource for anyone who's trying something for the first time. If you do things differently from another lab member, talk about it on the discussions page.
Online Science Resources
http://www.expasy.org/ The proteomics Server. I tend to use one of their tools for DNA-Protein translation purposes.
http://workbench.sdsc.edu/ Here you can collect your sequences and do alignments and lots of other things.
http://www.idtdna.com/SciTools/SciTools.aspx Primer design, oligo analyzer and much more.
http://www.ncbi.nlm.nih.gov/gquery/gquery.fcgi?itool=toolbar Home of lots of things. Also BLAST.
http://bioinformatics.org/sms/ Lots of DNA-Protein tools including a protein mass calculator.
Tips and Tricks
Cloning
If you need to separate large DNA strands from very small ones (>40bp), say when you’re preparing a plasmid to receive an insert, or your PCR product that you’ve cut with restriction enzymes, you can use Qiagen’s PCR cleanup kit rather than doing a gel purification. You’ll appreciate the increased yield and the time savings. I tried this today, and it works rather well. The cleanup procedure takes about 5 minutes (gel purification takes the better part of a day!).
If you need to screen a large number of transformants, you can do it by "Colony PCR". Transfer your colonies to a numbered gridplate with a yellow tip, and then stick the tip in a PCR tube that has 20ul of H2O in it. Remove the tip, boil the samples for 5 minutes and spin. Use 2ul as a template in a normal PCR.
Restriction enzymes
If you're having problems finding identical restriction sites for subcloning, look for sites that have compatible sticky overhangs. BamHI and BglII come to mind. There are lots more. The NEB catalog is your friend!
If you're really in trouble, and can only use an enzyme that also cuts in your insert, you can try your hand at a partial digest. Use a limiting dilution of the enzyme for a set period of time. Then gel purify the fragment that is the desired size.
You can destroy a site, change it into a different site, or introcuce a (frame shift) mutation by cutting this site, and then either fill in the 5' sticky overhangs, or chewing them up (3') with T4 DNA Polymerase, and ligating it back together.
Plasmid preps
The Marligen kits that we have use a precipitation in the final step. Be aware that after your 70% ethanol wash, the pellet does not always stick to the tube very well. This is usually where things go wrong.
Protocols
Hanahan protocol for making competent cells and transformation
1. Grow O.N. culture in LB not shaking.
2. Sub-culture cells 1:25 in SOB. Grow to OD600 ~ 0.5.
3. Place on ice 10-15 min. and transfer to cold centrifuge tubes 4. Spin cells at 5000 x g for 5 min.
5. Resuspend in 1/3 V TFB. Mix with GENTLE vortexing. Incubate on ice for 10-15 min.
6. Spin cells at 5000 x g for 5 min.
7. Resuspend in 1/12 V TFB. Mix with GENTLE vortexing.
8. Add DMSO to a final concentration of 3.5%. Incubate on ice for 5 min.
9. Add 2.5M DTT to a final concentration of 75mM. Incubate on ice for 5 min.
10. Add DMSO to a final concentration of 7%. Incubate on ice for 5 min.
11. Aliquot 200 ul in cold tubes. (six Pasteur pipet drops) Store at -80ºC.
12. Add DNA in a volume < 10ul.
13. Incubate on ice for 30 min.
14. Incubate at 42ºC for 90 sec.
15. Place on ice and add 800 ul SOC.
16. Incubate at 37ºC for 45 min. with SLOW shaking.
17. Spin cells for 1 min.
18. Remove 800 ul.
19. Resuspend cells in remaining 200 ul.
20. Plate 100 ul.
21. Transformation efficiency should be around 1*108.
SOB (200ml)
• Tryptone 4g
• Yeast extract 1g
• NaCl 116mg
• KCl 36mg
• 10 N NaOH 80ul
• H2O 196ml
After autoclaving, add 2ml sterile 1M MgCl2 and 2ml Sterile 1M MgSO4.
SOC
• 90 ul 40% Dextrose (Glucose) per 5 ml SOB.
TFB (200ml)
• MES 0.39g
• MnCl2*4H2O 1.78g
• CaCl2*2H2O 0.29g
• KCl 1.49g
• Hexamine Co(III)Cl2 0.16g
Add MES first, then adjust pH to 6.3 with 1N KOH. Then add remaining ingredients. Store TFB at 4ºC protected from light.
Making a glycerol stock
1. Inoculate 5ml of LB with a touch of your colony. (use a sterile yellow tip.)
2. Grow O.N. not shaking.
3. The next morning inoculate 3ml of fresh LB with 1ml of your culture.
4. Grow shaking for about 3-4 hours.
5. Add 1ml of 80% glycerol in LB.
6. Store 1ml aliquots at -80°C. (spread over multiple freezers in case of failure.)
Keep these stocks frozen at all times. If you need to grow some of these, scrape a little of with a sterile yellow tip. I don't really know how long these will last, but it is probably a good idea to check them once every 6 months, and replace them every two years.
