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Preprint

SFB colonization

DE Daria Esterházy
DM Daniel Mucida

Last updated date: Jun 28, 2021 Views: 848 Forks: 0

original An abbreviated version of this protocol was published in Nature
Compartmentalized gut lymph node drainage dictates adaptive immune responses
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1. fecal DNA extraction kit: Zymo research, Cat. No. D6010, use according to manufacturer's protocol

2. 16S primers:

SFB:

SFB736F: GACGCTGAGGCATGAGAGCAT

SFB844R: GACGGCACGGATTGTTATTCA

 

UNIVERSAL(control):

UNIF340: ACTCCTACGGGAGGCAGCAGT

UNIR514: ATTACCGCGGCTGCTGGC

 

 

 

Program for q-PCR:

95C 3’ (or 10 min if Applied Bio SYBR Green mix)

40 cycles of 10’’ at 95C and 45’’ at 62C.

 

per sample:

1 ul DNA

10 ul SYBR mix

0.4 ul primer mix at 20 nM each

8.6 ul water

 

The cycle at which SFB comes up depends on the machine. If you do not have germ free mice use a Jax mouse fecal pellet or, less ideal, antibiotics treated mouse fecal pellet as negative control. The universal 16S can be used as a reference/normalization (for non-antibiotic treated mice).

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How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Esterházy, D and Mucida, D(2021). SFB colonization. Bio-protocol Preprint. bio-protocol.org/prep1226.
  2. Esterházy, D., Canesso, M. C., Mesin, L., Muller, P. A., Castro, T. B. D., Lockhart, A., ElJalby, M., Faria, A. M. and Mucida, D.(2019). Compartmentalized gut lymph node drainage dictates adaptive immune responses. Nature 569(7754). DOI: 10.1038/s41586-019-1125-3

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