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1. Fragment length can be between 301-500 nt (including constant flanks).
2. The total length of the fragment cannot exceed 500 nucleotides including constant flanks which will be used for PCR amplification. Flanking sequences must be at least 20-25 bps in length, which will allow for up to 460 bps of variable coding region. 
3. The variation of fragment lengths from shortest to longest should not exceed 80 bp (e.g. if the longest fragment is 400 bp, the shortest fragment should be > 320 bp).

Yes, we can add standard Twist flanks to your fragments that can be used with restriction cloning.

No, we can only synthesize sequences containing the four standard bases A, C, G and T.  The use of degenerate bases (R, Y, S, W, K, M, B, D, H, V, N) are not allowed. However, if you would like to have all four bases (or a subset of bases) at a given position in a fragment, you can design separate fragments, each with a different base at the site of interest.

There are no complexity requirements for MGF. However, some features may cause suboptimal results:

1. Features like direct repeats and conserved sequences shared across all variants may result in some recombination during amplification.
2. Long homopolymers (>20bp) may result in some drop out during amplification.
3. Strong hairpins may result in poor amplification, which may lead to underrepresentation or dropout of those variants.

Pricing depends on the complexity tier (Pool Size) and the fragment length tier (301-350nt, 351-400nt, 401-450nt and 451-500nt).

The turnaround time for Multiplexed Gene Fragments is 8-12 business days.

Please consider the standard rules for primer design:

1. Length of 20-25 bp
2. 35-70% G/C content
3. Melting temperature (Tm) of 55-60°C
4. Primer pairs should have a Tm within 5°C of each other
5. Primer pairs should not have complementary regions
6. Avoid internal primer binding sites in the variable region

1. Capillary electrophoresis is performed on the final pool to determine purity of the sample. At least 90% of the molecules in the pool must fall within the expected length.
2. Fluorescence-based quantification is performed on the final sample to ensure that there is at least 200ng of material.
3. Twist does not perform any sequencing on the pool, and does not guarantee quality in terms of representation or error rate. However, we have seen during development that most pools have a 90/10 percentile of < 3 and an average error rate of ~1:2,000.

No, all fragments in the pool must have the same flank sequences. Multiple sets of flanks can be accomplished by ordering multiple pools. Alternatively, you can design a single pool with internal flanks that are different and external flanks that are universal for Twist to use for amplification. You may then amplify out the subpools; although this does come with inherent risk of skewing uniformity and/or running out of template material.

Step 1: Download the MGF Submission Form and fill out all fields in the form for your MGF design.  

Step 2: Upload the completed submission form here.  

If you have any questions or issues, please contact us via email (customersupport@twistbioscience.com) or chat.

1. Fragment length can be between 301-500 nt (including constant flanks).
2. The total length of the fragment cannot exceed 500 nucleotides including constant flanks which will be used for PCR amplification. Flanking sequences must be at least 20-25 bps in length, which will allow for up to 460 bps of variable coding region. 
3. The variation of fragment lengths from shortest to longest should not exceed 80 bp (e.g. if the longest fragment is 400 bp, the shortest fragment should be > 320 bp).

Yes, we can add standard Twist flanks to your fragments that can be used with restriction cloning.

No, we can only synthesize sequences containing the four standard bases A, C, G and T.  The use of degenerate bases (R, Y, S, W, K, M, B, D, H, V, N) are not allowed. However, if you would like to have all four bases (or a subset of bases) at a given position in a fragment, you can design separate fragments, each with a different base at the site of interest.

There are no complexity requirements for MGF. However, some features may cause suboptimal results:

1. Features like direct repeats and conserved sequences shared across all variants may result in some recombination during amplification.
2. Long homopolymers (>20bp) may result in some drop out during amplification.
3. Strong hairpins may result in poor amplification, which may lead to underrepresentation or dropout of those variants.

Pricing depends on the complexity tier (Pool Size) and the fragment length tier (301-350nt, 351-400nt, 401-450nt and 451-500nt).

The turnaround time for Multiplexed Gene Fragments is 8-12 business days.

Please consider the standard rules for primer design:

1. Length of 20-25 bp
2. 35-70% G/C content
3. Melting temperature (Tm) of 55-60°C
4. Primer pairs should have a Tm within 5°C of each other
5. Primer pairs should not have complementary regions
6. Avoid internal primer binding sites in the variable region

1. Capillary electrophoresis is performed on the final pool to determine purity of the sample. At least 90% of the molecules in the pool must fall within the expected length.
2. Fluorescence-based quantification is performed on the final sample to ensure that there is at least 200ng of material.
3. Twist does not perform any sequencing on the pool, and does not guarantee quality in terms of representation or error rate. However, we have seen during development that most pools have a 90/10 percentile of < 3 and an average error rate of ~1:2,000.

No, all fragments in the pool must have the same flank sequences. Multiple sets of flanks can be accomplished by ordering multiple pools. Alternatively, you can design a single pool with internal flanks that are different and external flanks that are universal for Twist to use for amplification. You may then amplify out the subpools; although this does come with inherent risk of skewing uniformity and/or running out of template material.

Step 1: Download the MGF Submission Form and fill out all fields in the form for your MGF design.  

Step 2: Upload the completed submission form here.  

If you have any questions or issues, please contact us via email (customersupport@twistbioscience.com) or chat.