View source for Team:UCLA/Notebook/Spider Silk Genetics/6 July 2015

{{Template: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.
{| class="wikitable"
! style="font-weight: bold;" | 
! style="font-weight: bold;" | 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
|-
| style="font-weight: bold;" | Total
| style="font-weight: bold;" | 25
|}
*NEB Tm calculator gives 65 C for annealing, based on the overlap between the forward and reverse.
*Test 63 C and 66 C.
{| class="wikitable"
| 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.
[[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.

==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.

#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
#*50 ng 2AB
#*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.