Team:Penn/Notebook

University of Pennsylvania iGEM

Content Expander

expanding content since 2014

robsawyer.me

Colony PCR


Protocol: Template Preparation 

  1. First, one must have a monoclonal E Coli colony source. This may be one of the following three things:
    1. A glycerol stock made from a SINGLE colony on an agar plate
    2. An overnight culture in LB media (+ antibiotics if appropriate) started from a SINGLE colony on an agar plate
    3. A SINGLE colony on an agar plate
  2. Label a sterile eppendorf with the strain number and assign a unique colony number for each colony picked. Pipet 20 uL of sterile water into the tube. If the e coli is coming from sources (1) or (3), use a clean, sterile pipet tip to scrape a tiny bit of bacteria (even the very smallest amount is okay) and then pipet up and down into the waiting 20 uL. If the e coli is coming from source (2), pipet 1 uL of the LB culture into the water. Mix well.
  3. This water/colony mixture will be the “template” for your PCR reaction. Next, set up your reaction mixture and run the cycler protocol. Follow this up by running a gel to examine the PCR amplicon lengths.


Reaction Preparation 

  1. 1 uL Template
  2. 10 uL 2x Taq Master Mix (Long term storage in freezer, will last one month at 4C)
  3. 1 uL FWD Primer (@ 10 uM)
  4. 1 uL REV Primer (@ 10 uM)
  5. 7 uL H2O


Cycler Protocol- The cycler protocol is fixed except for the extension time. Set the extension time for 1 min per kb of desired amplicon at 68C, plus a little bit of buffer time. For example, for a 2 kb amplicon, set the extension time to 2 min 30 seconds.

  1. 95ºC for 6 minutes
  2. 30x [95ºC for 30 sec, 55ºC for 30 sec, 68ºC for 1 min/kb amplicon]
  3. 68ºC for 20:00 min
  4. Hold at 4ºC

DNA Digestion


Reaction Mixture Preparation

  1. 5 uL 10X Digest Buffer
  2. 1 uL of each digestion enzyme up to maximum of 2.5 uL
  3. 2000 ng of desired cut fragment up to max volume of 50 - enzyme volume (cutsmart+digestion enzyme volumes)
  4. Fill with milliQ water to 50 ul


Digestion Protocol 

  1. Incubate the reaction at 37ºC for 1 hour.
  2. The digest buffer is specific to the enzyme and it stated on the enzyme product description - look it up on NEB’s webpage for each enzyme. It is generally “Cutsmart.” For digests with multiple enzymes, use NEB’s tool to identify the right buffer.
  3. Enzymes are stored in glycerol, but glycerol can inhibit digestion reactions. To keep the volume to 2.5 uL, use 1 uL of 2 enzymes. For three enzymes, consider using just 0.5 uL of the most active one or reducing the volume of all three to 0.8 uL.
  4. You want there to be at least 2000 ng of the fragment you want to isolate in the end. This is because DNA digestion products must be purified, a process that is only about 50% efficient. Isolating 2000 ng ensures you have enough DNA in the end for cloning. To calculate the volume, use the DNA concentration and the relative fraction of the plasmid that is the fragment you want. For example: for a 10 kb plasmid at 1000 ng/uL with a 1kb fragment I want to cut out, I’d want to do the following calculation:

DNA Gel Extraction


Gel extraction: Follow the Qiagen QiaQuick DNA Gel Extraction protocol that accompanies their kits. Below are the essential steps:

  1. Add 3 volumes of QG buffer per 100 mg of agarose gel (i.e. add 450 uL of QG buffer for a 150 mg agarose gel chunk)
  2. Heat at 50ºC for ~10 minutes until the gel completely dissolves. Vortex to mix.
  3. Add 1 volume of isopropanol per 100 mg of agarose gel (i.e. add 150 uL of Isopropanol to a 150 mg agarose chunk dissolved in 450 uL of QG buffer)
  4. Vortex to mix.
  5. Add up to 700 uL isopropanol-QG-DNA-Agarose mixture to a gel extraction column (purple with lids). Spin down at 13000 rpm for 1 minute. Discard the flow through.
  6. Repeat step 5 until you have spun down all the dissolved gel you have
  7. Pipet 750 uL of PE buffer (**Make sure 200 proof Ethanol has been added) onto the column
  8. Spin down at 13000 rpm for 1 min. Discard the flow through.
  9. Air dry by spinning down at 13000 rpm for 1 min.
  10. Move the column to a fresh eppendorf tube. Pipet 35 uL of EB buffer above the column’s membrane - careful not to puncture it!
  11. Let the EB buffer sit on the membrane for 1 min.
  12. Spin at 13000 rpm for 1 min to elute the purified DNA into the eppendorf tube.
  13. Consider measuring concentration on the nanodrop (see separate protocol)


 

DNA Gel Extraction

It is best to follow the QiaQuick PCR Purification protocol supplied with their kit. Below are the essential steps: (All buffers and columns are supplied with the QiaQuick PCR purification kit.)

  1. Add 5 volumes of PB (binding) buffer to the DNA sample you’d like to purify. For example, if you have a 50 uL completed PCR reaction to cleanup, start by adding 250 uL of PB buffer and mixing well.
  2. Add the PB + DNA mixture to a PCR cleanup column (purple columns, same as gel extraction). Spin at 13000 rpm for 1 min. Discard the flow through.
  3. Pipet 750 uL of PE buffer onto the column. Spin at 13000 rpm for 1 min. Discard the flow through.
  4. Air dry the column by spinning at 13000 rpm for 1 min. Transfer the dried column to a fresh eppendorf tube.
  5. Add 35 uL of EB buffer to the column and allow it to stand for 1 min.
  6. To elute, spin at 13000 rpm for 1 min. The solution that flows into the eppendorf collection tube is your purified DNA. Consider measuring its concentration with the nanodrop.