The myTXTL Pro Kit is best for rebooting bacteriophage and some modifications to the reaction mix may be needed for rebooting your phage, please refer to the manual for detailed suggestions on phage rebooting.

An additional application for the myTXTL Pro kit is that it can be used to reboot bacteriophage from a genome input. Several phages have been shown to replicate in the myTXTL system and they generally benefit from the following modifications:

1) The addition of 0.3 mM each of additional dNTPs (for replication of DNA genomes) and 0.5-4% PEG 8000 can be helpful for optimal production of phage. These components along with your genome, 9 uL of Pro Master Mix, and water if needed should add up to 12 uL and be assembled in a 1.5/2 mL tube. Larger reaction volumes may require shaking as described in the manual for larger scale reactions. Incubate overnight as in our protocol.
2) You may also need to optimize the phage genome concentration if it is not already published. 0.25 nM has worked well for T7 but T4 was optimal at 1 nM. In part this is dependent on the quality of the genome. It is extremely important that the phage genome is of very high purity, free of contaminants and of high integrity.
3) Commercially available T7 phage genomic DNA can be used directly in the Pro Master Mix as a positive control (we have used DNA from BocaScientific:

A standard protocol for chemical transformation usually produces E. coli cells with a sufficient competency for plasmid intake. We recommend following the procedure described in Sambrook et al. 1989.

  1. Master mix inactivation due to improper storage. The myTXTL master mix must be stored at -80°C and number of freeze-thaw cycles should be minimized.
  2. Improper reaction setup, Please review the recommendations to set up a myTXTL reaction in the current myTXTL manual.
  3. Contamination of myTXTL reaction with nuclease. To avoid nuclease contamination, wear gloves and use nuclease-free water, sterilized tips and tubes. Use deionized, nuclease-free, “molecular grade” water for myTXTL reaction setup.

Batch-to-batch variation can cause varying levels of TXTL inhibitor contamination present in the plasmid solution. Please follow our recommendations on how to prepare template plasmid DNA for TXTL reactions in the current myTXTL manual.

If you have started your project with plasmid templates for which you purchased the myTXTL Sigma 70 Master Mix, but now you want to try out linear DNA templates with the myTXTL Sigma 70 Master Mix you still have available in your lab. A simple addition of myTXTL GamS Protein to the myTXTL Sigma 70 Master Mix significantly boosts protein output from linear DNA templates in this master mix. For future work with linear DNA templates we would recommend the myTXTL Linear DNA Expression kit or myTXTL T7 Expression kit as both use the myTXTL Linear DNA Master Mix.

The kit used depends on the phage’s genome type, if it is linear DNA, we recommend the myTXTL Linear DNA kit or myTXTL T7 Expression Kit if you need to express accessory proteins. If circular DNA or RNA, any of our kits could be used, keeping in mind that non-E. coli phages may require the addition of their host’s primary transcription factor (SigA/Sigma70) or other accessory proteins to enable replication (see Emslander et al. 2022).

Unfortunately, not. However, studies have shown that supplementing cell-free systems with mixtures of reduced (GSH) and oxidized glutathione (GSSG), disulfide bond isomerase C (DsbC), protein disulfide isomerase (PDI) and/or chaperones (e.g. DnaK, DnaJ, GroEL, GroES) can promote the formation of disulfide bonds. In addition, pretreatment with iodoacetamide (IAM) to inactivate endogenous reductases which are present in the cell extract might also help (Review Article: Stech M & Kubick S, Antibodies 2015, 4, 12-33).

Yes, although it is not optimized or recommended for linear DNA templates. If using linear DNA with Sigma 70 MM, enhanced protein yields can be achieved by supplementing the master mix with our nuclease inhibitor GamS.

We always recommend keeping the thawing-and-freezing cycles as low as possible which might require aliquoting the original sample. However, myTXTL GamS Protein can be subjected to at least five thawing-and-freezing cycles.