BACE - DNA combination and Ship

Some samples still have poor yields, so combine samples and reconcentrate

Followed protocol for Amicon Pro Purification System:

 MCBA15 004 - combine 4.1 and 4.2 from 2/25

 MCBA15 007 - combine from both extraction days

 MCBA15 015 - combine from both extraction days

 MCBA15 017 - combine from both extraction days

 MCBA15 019 - combine 19.1 and 19.2 from 2/25

 MMLR15 020 - combine from both extraction days

Quantified with Qubit on BioTek at 485/530 nM

Sample ID     Concentration (ng/uL)   Volume (uL)

MCBA15 004           20.8                 95

MCBA15 007            7.7                 90

MCBA15 015           34.9                100

MCBA15 017*          81.0                100 

MCBA15 019           11.0                 90

MMLR15 020            4.9                 95

 *split in two  

Shipped out sample on 3/3/15 (due to weather):

Sample ID        Total DNA (ng)

MCBA15 004          1976.0

MCBA15 007           693.0

MMLR15 010*          672.0

MMLR15 011*          616.0

MCBA15 015          3490.0

MCBA15 017          3645.0

MCBA15 019           990.0

Samples delivered and received 3/4/15 - email from Michael

_____________________________________

Samples not sent out due to poor yields:

MMLR15 018 - slow growing

MCBA15 021 - Frigoribacterium; did not redo

MMLR15 022 - Frigoribacterium; did not redo

BACE - DNA Extraction Pt II

Try and extract DNA from samples that I could not get enough DNA from on 2/17

   ***for samples with really poor yields from last time, extracted two sets

Need more Lysozyme - 10 mg/mL in 60 uL x 20 samples

   TEN Buffer: 

      40 mM Tris-HCl ph=7.5

      1 mM EDTA ph=8.0

      150 mM NaCl

      Stock Solutions:      

      400 mM Tris-HCl = 6.30 g in 100 mL dH2O

      100 mM EDTA = 2.92 g in 100 mL dH2O

      300 mM NaCl = 1.75 g in 100 mL dH2O

      --> 1500 uL TEN Buffer = 750 uL NaCl + 15 uL EDTA + 150 uL Tris-HCl + 585 uL ddH2O

   Add 1 mL TEN Buffer + 10 mg Lysozyme = 10 mg/mL

Followed Promega Wizard DNA Purification Kit Protocol for gram-positive bacteria

   EXCEPT:

      Added 2 mL of liquid grown culture

      Added 10 mg/mL of 60 uL + 60 uL ddH2O = 120 uL

      Added 60 uL of Rehydration Solution

Quantified with Qubit kit on BioTek at 485/530 nM

Sample ID     Concentration (ng/uL)

MCBA15 004.1      7.9

MCBA15 004.2     20.8

MCBA15 007        4.9

MMLR15 010        0.0 - probably lost pellet 

MCBA15 015       10.7

MCBA15 017.1      8.7

MCBA15 017.2      0.3

MMLR15 018.1      1.0 - grows slow, not much input

MMLR15 018.2      3.1 - grows slow, not much input

MCBA15 019.1      7.5

MCBA15 019.2     13.5

MMLR15 020       16.6

BACE - DNA Extractions and Shipment

Shipped out samples to MIT - Martin Polz and Michael Cutler

Curtobacterium samples (n=11) sent on 2/17/15:

Sample ID      Total DNA (ng)
MCBA15 001         981.0
MMLR15 002        1254.2
MCBA15 003         893.8
MCBA15 005         953.9
MMLR15 006         943.6
MCBA15 008        1657.8
MCBA15 009         922.6
MCBA15 012         962.3
MCBA15 013        1240.4
MMLR15 014        1153.8
MCBA15 016         756.7

Samples Received on 2/19/15

BACE - DNA Extraction

Followed Promega DNA Extraction Kit Protocol

Results were better than Spin Column method, but still not great for some samples.