Difference between revisions of "Team:UCLA/Notebook/Spider Silk Genetics/6 July 2015"

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==Results==
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===Results===
 
*Cast 1.5 % TAE gel, used 2 uL of 50 bp ladder to visualize.
 
*Cast 1.5 % TAE gel, used 2 uL of 50 bp ladder to visualize.
 
[[File:7 6 2015.jpg|none|thumb|500px|'''Fig. 1''' Assembly of MaSp2 sequencing core. The size of the expected product is 84 bp.]]
 
[[File:7 6 2015.jpg|none|thumb|500px|'''Fig. 1''' Assembly of MaSp2 sequencing core. The size of the expected product is 84 bp.]]
 
*The assembly was successful.
 
*The assembly was successful.
 +
 +
==ICA using M-270 Beads==
 +
*The [https://www.lifetechnologies.com/order/catalog/product/21344 Streptavidin M-270 Dynabeads] came in today. We will test ligation of a 3-mer using these beads to see if we get a different result compared to using [https://www.lifetechnologies.com/order/catalog/product/21344 Magnabind Streptavidin] beads.
 +
*Ligations were performed in 10 uL reactions using T7 ligase (Enzymatics), 50 ng of the relevant DNA, and 1 uL of the relevant 5 uM capping oligo.
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 +
#5 uL of beads were used.
 +
#*Washed twice using 2x BW buffer.
 +
#Resuspend washed beads in 5 uL 2x BW buffer, 1 uL of 5 uM initiator, and 4 uL ddH2O.
 +
#Rotate at RT for 45 minutes.
 +
#Wash twice using 0.5x BW buffer.
 +
#Add ligation mix
 +
#*5 uL 2x T7 ligase buffer
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#*50 ng 2AB
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#*0.5 uL TL ligase
 +
#*ddH2O to 10 uL
 +
#Repeat ligations and washes as needed.
 +
#Final wash using ddH2O.
 +
#Elute in 15 uL 0.01% Tween-20 at 95 C for 3 min.
 +
#Saved Elution.

Latest revision as of 16:18, 8 July 2015

iGEM UCLA




7/6/2015

MaSp2 Sequencing Core Assembly

  • PCA for MaSp2 Sequencing Core to be used for sequencing longer MaSp constructs.
  • Used the MaSp2 Seq Core 1-F (D-05) and MaSp Seq Core 1-R (E-05) to create full length MaSp sequencing core.
Volume (uL)
5x Q5 Buffer 5
10 mM dNTPs 0.5
10 uM For (D-05) 1.25
10 uM Rev (E-05) 1.25
5x GC Enhancer 5
ddH2O 11.75
Q5 Polymerase 0.25
Total 25
  • NEB Tm calculator gives 65 C for annealing, based on the overlap between the forward and reverse.
  • Test 63 C and 66 C.
98 C 30 sec
98 C 10 sec
63, 66 C 15 sec
72 C 10 sec
repeat from step 2 25 x
72 C 2 min
12 C hold

Results

  • Cast 1.5 % TAE gel, used 2 uL of 50 bp ladder to visualize.
Fig. 1 Assembly of MaSp2 sequencing core. The size of the expected product is 84 bp.
  • The assembly was successful.

ICA using M-270 Beads

  • The Streptavidin M-270 Dynabeads came in today. We will test ligation of a 3-mer using these beads to see if we get a different result compared to using Magnabind Streptavidin beads.
  • Ligations were performed in 10 uL reactions using T7 ligase (Enzymatics), 50 ng of the relevant DNA, and 1 uL of the relevant 5 uM capping oligo.
  1. 5 uL of beads were used.
    • Washed twice using 2x BW buffer.
  2. Resuspend washed beads in 5 uL 2x BW buffer, 1 uL of 5 uM initiator, and 4 uL ddH2O.
  3. Rotate at RT for 45 minutes.
  4. Wash twice using 0.5x BW buffer.
  5. Add ligation mix
    • 5 uL 2x T7 ligase buffer
    • 50 ng 2AB
    • 0.5 uL TL ligase
    • ddH2O to 10 uL
  6. Repeat ligations and washes as needed.
  7. Final wash using ddH2O.
  8. Elute in 15 uL 0.01% Tween-20 at 95 C for 3 min.
  9. Saved Elution.