Prbbbb:fusion assembly v1

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Current revision (04:48, 4 May 2010) (view source)
(Restriction: correct sensitivity of different restriction mixes to Tris-HCl buffer)
 
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Note:
Note:
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* The restriction mixes below are calculated for 10 µl final volume for the digest -- 2 µl restriction mix + 8 µl DNA. We do '''not''' add any further water to the reaction but instead use a standard (rather low) concentration of DNA. Especially in case of restriction A, it is critical that the DNA sample has been eluted in water, and not in Elution buffer (TRIS) which would otherwise interfere with the restriction buffer.
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* The restriction mixes below are calculated for 10 µl final volume for the digest -- 2 µl restriction mix + 8 µl DNA. We do '''not''' add any further water to the reaction but instead use a standard (rather low) concentration of DNA. Especially in case of restriction C, it is critical that the DNA sample has been eluted in water, and not in Elution buffer (TRIS) which would otherwise interfere with the restriction buffer.  
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** Test-restrictions using DNA in Tris-HCl (Qiagen elution buffer) failed in case of mix C and were somewhat reduced for mix A
* Complete digestion is critical for a low background -- at least initially, check your digests on a gel!
* Complete digestion is critical for a low background -- at least initially, check your digests on a gel!
* The digest volume can be reduced further to 5 µl (4 µl DNA + 1 µl restriction mix). This leaves no sample for a gel though.
* The digest volume can be reduced further to 5 µl (4 µl DNA + 1 µl restriction mix). This leaves no sample for a gel though.

Current revision

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graphical overview

Contents

Overview

This variant of the classic 3A assembly protocol minimizes pipetting steps and the amount of DNA used. The trick is to start from standard DNA dilutions so that DNA and restriction solutions can be mixed without any further water. Most of this protocol can be performed in a 96-well pcr plate on 12 or 24 assemblies in parallel.

Graphical overview as high resolution PDF

The description below uses the RFC 25 (Fusion) standard for assembly but applies equally to the classic assembly. Just replace enzymes and buffers in restriction mix A and B.

Materials

Procedure

Restriction

Note:

  • The restriction mixes below are calculated for 10 µl final volume for the digest -- 2 µl restriction mix + 8 µl DNA. We do not add any further water to the reaction but instead use a standard (rather low) concentration of DNA. Especially in case of restriction C, it is critical that the DNA sample has been eluted in water, and not in Elution buffer (TRIS) which would otherwise interfere with the restriction buffer.
    • Test-restrictions using DNA in Tris-HCl (Qiagen elution buffer) failed in case of mix C and were somewhat reduced for mix A
  • Complete digestion is critical for a low background -- at least initially, check your digests on a gel!
  • The digest volume can be reduced further to 5 µl (4 µl DNA + 1 µl restriction mix). This leaves no sample for a gel though.
  • The whole protocol can be performed in a 96-well PCR plate (see below)

restriction mix A (5x concentrated)

1 µl, single reaction 20 µl 60 µl 120 µl
H2O 0.25µl 5 15 30
10 x NEBuffer 1 0.5µl 10 30 60
EcoRI 20U/µl 0.05µl 1 3 6
AgeI 5U/µl 0.2µl 4 12 24

Thanks to the high Glycerol content, the restriction mix can be stored at -20 for a couple of weeks.

restriction mix B (5x concentrated)

1 µl, single reaction 20 µl 60 µl 120 µl
H2O 0.2µl 4 12 24
10 x NEBuffer 4 0.5µl 10 30 60
100 x BSA 0.05 1 3 6
PstI 20U/µl 0.08µl 1.6 4.8 9.6
NgoMI 10U/µl 0.17µl 3.4 10.2 20.4

Thanks to the high Glycerol content, the restriction mix can be stored at -20 for a couple of weeks.

restriction mix C (5x concentrated)

1 µl, single reaction 20 µl 60 µl 120 µl
H2O 0.2µl 4 12 24
10 x NEBuffer EcoRI 0.5µl 10 30 60
100 x BSA 0.05 1 3 6
EcoRI 20U/µl 0.125µl 2.5 7.5 15
PstI 20U/µl 0.125µl 2.5 7.5 15

Thanks to the high Glycerol content, the restriction mix can be stored at -20 for a couple of weeks.

  1. mix 8 µl part A [50 ng/µl] with 2 µl restriction A
  2. mix 8 µl part B [50 ng/µl] with 2 µl restriction B
  3. mix 8 µl vector [25 ng/µl] with 2 µl restriction C
    • (better: prepare pre-digested, dephosphorylated stock by vector PCR)
  4. incubate for 2h @ 37°C
  5. heat inactivate 20' @ 80°C

Ligation

ligation mix (2x concentrated)

10 µl, single reaction 150 µl 600 µl µl
H2O 7µl 105 420
10 x T4 buffer 2µl 30 120
T4 DNA Ligase 1 15 60
  1. mix 4 µl part A digest + 4 µl part B digest + 2 µl vector digest
  2. add (as last component!) 10 µl ligation mix (2x)
  3. incubate 1h @ 16°C; 10' @ 65deg;C
  4. use 2 µl for transformation

Performing assemblies on a 96-well plate

  1. multi-dispense restriction mix A into row A
  2. multi-dispense restriction mix B into row B
  3. add 8 µl parts DNA [50ng/µl] to rows A and B
    • Example: you want to couple part 1 to part 2 and, in parallel, part x to part y. Put part 1 into A1, part 2 into B1, part x into A2, part y into B2
  4. program & run restriction + heat shock on a PCR device
  5. use multi-channel pipette to copy 4 µl from A and B into row D
  6. add 2 µl pre-digested vector backbone into each well of D
    • except if there is an insert-only control -- add water instead
  7. multi-dispense 10µl ligation mix into D
  8. seal plate and run ligation protocol on PCR device
  9. put plate on ice
  10. pipette (gently!) 12 µl competent cells into each well of row F
  11. copy 2 µl from ligation row D into cell row F
  12. run first half of transformation (20'@4C; 30"@42C; 10'@4C) on PCR device
  13. dilute 12 µl cells into 200 µl S.O.C. medium in a sterile 96 x 1.5 ml deepwell plate
  14. seal the deepwell plate and incubate 45 min @ 37°C with vigorous shaking
  15. plate 200 µl on LB Agar with the appropriate antibiotic (select for target vector)
    • The Knight lab robotic protocol uses serial dilution instead of plating: robot assembly
  16. incubate over night @ 37°C, and continue with colony screening protocol

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.


References

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