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Protocols

Making cells competent

Transformations

Standard transformation procedure

• Remove competent cells from -80, let thaw for 10 min on ice and aliquot in 50 ul amounts.
• add 2-5 ul of vector, usually in H2O, to 50 ul cells, no mixing by pipet due to shear induction.
• keep on ice for 20 minutes (vector spreading through volume)
• heat shock (42°C) for 45 seconds
• keep on ice for 2 minutes
• add 200 ul SOC, put on 37°C for 1 hour or longer with agitation.
• plate out 250 ul on appropriate antibiotics.

Restrictions

Try to do a restriction in a relatively large volume. As a rule of thumb, use a volume of 50 ul / 500 ng DNA.

• Calculate the amount of DNA you want to use
• add H2O
• add 10 x H buffer (Roche)
• add your calculated amount of DNA
• add 0.5 ul of each enzyme. Keep in mind 0.5 ul = 5 U, where 1 U is defined as the amount of enzyme cutting 1000 ng of DNA / hour, so for extremely large amounts of DNA adjust this.
• keep on 37°C for 2-3 hours.

Purifying small DNA parts

Protocol found on OpenWetWare

This protocol is for a simple ethanol precipitation of small fragments. This protocol was used to (partially) purify a DNA fragment containing a ribosome binding site (~40 bp) during 3A assembly]. The fragment was generated via restriction digest and it was used in a ligation reaction. Note that this protocol simply concentrates your sample and removes enough salts/enzymes for ligation to be successful. All DNA fragments from your digest will still be present in your pellet. These residual DNA fragments do not matter for 3A assembly which selects against incorrect ligation products.

Materials

• Absolute Ethanol (100% = 200 proof)
• 95% ethanol
• Tabletop centrifuge
• -80°C freezer

Procedure

1. Add 2 volumes ice cold absolute ethanol to sample.
   Generally the sample is in a 1.5 mL eppendorf tube. I recommend storing the absolute ethanol at -20°C.
2. Incubate 1 hr at -80°C.
   The long incubation time is critical for small fragments.
3. Centrifuge for 30 minutes at 0°C at maximum speed (generally >10000 g at least).
4. Remove supernatant.
5. Wash with 750-1000 μL room-temperature 95% ethanol.
   Another critical step for small fragments under 200 base pairs. Generally washing involves adding the ethanol and inverting several times.
6. Centrifuge for 10 minutes at 4°C at maximum speed (generally >10000 g at least).
7. Let air dry on benchtop.
   I generally let the pellet air dry completely such that it becomes white so that all residual ethanol is eliminated.
8. Resuspend in an appropriate volume of H₂O.
   Many protocols recommend resuspending in 10 mM Tris-HCl or TE. The advantage of TE is that EDTA chelates magnesium ions which makes it more difficult for residual DNases to degrade the DNA. I generally prefer H₂O and don't seem to experience problems of this sort. If you plan to ultimately use electroporation to transform your DNA then resuspending in H₂O has the advantage of keeping the salt content of your ligation reaction down.

Ligation

First make sure you have purified the DNA after restriction. Ligation should be in a small volume (we usually use 15 ul), so elute your DNA from the column in a small volume/high concentration.

• add H2O
• add 10 x ligation buffer
• add backbone and insert (theoretically in a 1:3 or 1:4 ratio, for 3A assembly it seemed to work at 1:1 ratios, possibly even better). DNA amounts added are at least 50 ng of the backbone and if possible 100-150 ng of the insert DNA (including it's backbone).
• add 1 ul of T4 Ligase.
• keep the reaction at 16ºC for at least 2 hours, but o/n is preferable.
• if used for transformation, all DNA can be added to competent cells, or if you want to analyze it on gel, keep 5 ul.

PCR

Colony PCR

• Make biobrick mastermix, containing per sample:
  • 12.5 ul Taq mastermix
  • 2.5 ul 10x forward biobrick primer
  • 2.5 ul 10x reverse biobrick primer
  • 7.5 ul H2O
• Put 25 ul in the PCR tubes.
• With a toothpick or pipet point, touch a colony and stir it through the fluid
• Run the iGEM colpcr program (to be added later)

PCR using Taq Mastermix

Contents of the PCR mix is the for a large part the same as mentioned above for the Colony PCR. Differences will be noted here. First, instead of biobrick primer, any primer of choice can be added, also 2.5ul if standard solution has a concentration of 10 pmol/ul. Also x ul template DNA from a sample is added, where x depends on the total concentration of DNA in the sample. Typically 50 to 100 ng of total DNA is added. 7.5 - x ul of H₂O is added to the mix.

PCR program is:
1. 5' @ 95ºC
2. 1' @ 95ºC
3. 1' @ annealing temperature of the primer
4. 1' @ 72ºC (1' is long enough for 1kb, longer times can be used if larger products are formed)
5. repeat steps 2-4 29x (total of 30 cycles, more can be added if necessary)
6. 5' @ 72ºC
7. forever @ 4ºC (PCR can be stopped and stored in the fridge at any time from this point on)

PCR using Pfx polymerase

Mastermix does not exist for the Pfx polymerase. This means the components have to be added seperately. The mix consists of:

• x ul template DNA (again 50 - 100 ng total)
• 5.0 ul 10x buffer
• 2.5 ul forward primer (10 pmol/ul)
• 2.5 ul reverse primer (10 pmol/ul)
• 0.2 ul Pfx
• 1.5 ul dNTP's (10 mM)
• 1.0 ul MgSO₄ (50 mM)
• 37.3-x ul H₂O

The PCR program looks the same as mentioned above for Taq polymerase, only difference is the elongation temperature in step 4. This is 68ºC for Pfx.

Gradient PCR

Gradient PCR is mainly used to determine the best annealing temperature for primers. This is done in this project with Taq polymerase mastermix, as this is cheaper than Pfx. However, as long as a PCR machine capable of making gradients is present, a gradient PCR can be performed with any polymerase. During the annealing step (step 3 in the taq mastermix protocol) every column in the PCR machine has a different temperature, going up from left to right. The range of the gradient can be installed manually, however the actual temperatures cannot (at least not in our machine). An example of PCR products put on gel after a gradient PCR can be seen in the lab notebook at the 20th of August, where gradients of 5ºC in 12 steps were tested for the atoB, idi and ispA primer pairs.

Touch Down PCR

DNA gels

• Take a flask of 0.8% up to 1.5% molten agarose from the 70oC stove.
• Pour a it in a taped gel tray.
• Add ca. 5 ul of SYBRSafe (depending on size gel)
• Add a comb and let the gel harden for ca. 15 minutes.
• Remove the comb and the tape and put the gel tray in an electrophoresis tray.
• Add enough 1x TBE to completely cover the gel.
• Add DNA loading buffer to your samples and load them.
• Let the gel run at a voltage between 60V and 120V, depending on desired resolution/time available.
• Visualize the DNA by putting it in the imager for taking a picture, or if you want to cut out your DNA, put it on the blue light emitter.

Luciferase Assays

Due to some protocols not working as desired, we've used various different ones. The one listed here is the latest, others can be found under the following links: 25th of September protocol; 15th of October protocol

Buffers