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