Paulsson:Plasmid replication

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Working with radioactivity in the Kirschner lab

do online safety test
talk to Mike Gage about getting on their permit
series code SKM
can order radionuclides through Harvard site using Kirschner Lab as the destination and permit holder, bubt James bills our grant (how was this made clear in purchasing process?)

Measuring DNA synthesis with radiolabeled thymidine

Before you begin:

1. Use a thy- strain (not thi-, that's thiamine not thymine),

or add 250 ug/ml deoxyadenosine to growth media to label strains that aren't thymine auxotrophs (Boyce and Setlow; BBA, 61:618, 1962)

2. [14C]-thymidine labeling before treatment (changing temp., adding drug, etc...) provides internal control for each sample pulse-labeled with [3H]-thymidine at time of treatment

3. CsCl equilibrium gradient will separate DNA base on GC content; addition of ethidium bromide will preferentially displace CsCl from change density of plasmid DNA - EtBr binding: linear and open circle (OC) > covalently closed circle (CCC)

4. sucrose velocity gradients can also be used, these will also separate open and closed plasmid DNA

5. The difference between thymine and thymidine: in case you forgot!, but what's the difference for labeling protocol? Perhaps thymidine is incorporated a little faster (doesn't require addition of deoxyribose), and is better for pulse studies like we want to do

New style

Labeling DNA

• Grow bugs in 10ml minimal medium, containing 250 ug/ml 2-deoxyadenosine, to a cell density of about 5x10E7/ml.
  
  • Optional: containing [methyl-14C]-thymidine (about 0.2 uCi/ml,; specific activity 50 mCi/mmol)
  • OR label for approx. 90' at 30 deg with [methyl-14C]-thymidine at 4 uCi/ml) (Hishimoto-Gotoh)
    
  • OR label for 6h at 25 deg with with [methyl-14C]-thymidine at 1 uCi/ml) (Eichenlaub)
• When cells are at OD600 ~ 0.1, pellet and resuspend in 10ml fresh media.
• Grow at 30° for 15 min in 50ml conical.
• Add [methyl-3H]-thymidine (10 uCi/ml; specific activity about 20 Ci/mmol)
  • Immediately transfer 1ml to microfuge tube on ice, containing 50ul 2M NaN3 and 50ul 2mg/ml thymidine.
  • Collect samples as above at time = 15' and 30'
• At 30', arrest plasmid replication by shifting temperature, or adding IPTG to 4mM.
  • Collect samples as above at time = 35', 40', 45', 50', 55' and 60'.
• Wash cells 2x with cold TE/NaN3 (????)

Sample collection: take 1ml of culture per time point (keep some concentrated & unlabelled cells to add to samples to achieve similar cell number per tube - cell density may affect pelleting efficiency?????

Plasmid isolation

• Perform standard QIAprep mini-prep. Elute 2x with 60ul EB (Elution Buffer) pre-warmed to 37°.
  • Set aside 10ul

• Remove background DNAs: linearize unwanted plasmids (e.g. pSC101 with Plac-copA) & digest linear plasmid and chromosomal DNA with Plasmid-Free exonuclease
  • 100 ul mini-prep
    
  • 12 ul 10x NEB buffer #4 OR 10x Plasmid-Free buffer (almost the same composition)
    
  • 6 ul 20mM ATP
  • 0.5 ul Restriction Enzyme (e.g. EcoNI)
  • 1.5 ul Plasmid-Free

120 ul total volume
Incubate at 37° for 60 minutes.

• Re-isolate plasmid DNA on QIAprep comlumn
  • Add 600 ul PBI to digest
  • Recover on QIAprep column
  • Elute with as much as possible for scintillation countingul EB (Elution Buffer) pre-warmed to 37°.

Re-isolation of plasmid DNA:

1. Use of a QIAprep column may be better than a QIAspin column which is designed for isolating smaller DNAs (restriction/PCR fragments).
2. Precipitation with TCA (trichloroacetate) may also be good - free nucleotides not recovered.

Scintillation counting

Need to wash filters to remove unincorporated label??? How to distinguish between counts from 14C and 3H: energy of beta-particle released is different and can be distinguished by the rate of scintillation events. Counters have multiple channels to do just this.

Fluid for Kirschner lab machine: ReadySafe (Beckman) P/N 141349 Vials: Beckman # 566740 Lids: Beckman # 587961 (500x)

References

1. Plasmids: A Practical Approach (Ch. 1, Kurt Nordstrom)
   
2. "Mutants of the mini-F plasmid pML31 thermosensitive in replication", Eichenlaub, R. JBact'79, v138(2):559-566.
   
3. "Mutations to temperature sensitivity in R plasmid pSC101", Hashimoto-Gotoh, T and Sekiguchi, M. JBact'77, v131(2):405-412.
   
4. "Degree or participation of exogenous thymidine in the overall DNA synthesis in E.coli", Daphna Rosenbaum-Oliver and Stephen Zamenhof. JBact'72, v110(2):585-5951.
   
5. "A simple method of increasing the incorporation of thymidine into the deoxyribonucleic acid of Eschericha coli", R.P. Boyce and R.B. Setlow. BBA'62, v61:618-620.